International Journal of Medical Laboratory

Glioma is the most prevalent and offensive type of intracranial brain tumor. It is the major hazardous brain tumor type in adults [1, 2]. For each type of glioma, some neoplasms span a broad spectrum of biological aggressiveness [3]. It can be estimated without treatment that the average life expectancy for patients is only 4–5 months [4]. Hence, the development of suitable strategies and recognition of novel pathways remain in urgent demand for the early diagnosis and treatment of glioma [5]. Based on the World Health Organization (WHO) classification, four grades are considered for gliomas: Grade I and Grade II are known as low-grade, and Grade III and IV are considered high-grade glioma [6]. Glioblastoma multiform (GBM) is the fourth grade of glioma and one of the most invasive brain tumors [7]. Malignant glioma is one of the most common causes of brain tumors and is considered one of the most dangerous cases in the nervous system indicates a relative scare but drastic health burden [8, 9].
On the other hand, although decades of rigorous research is available, the intricate biology of GBM is not entirely understood [10]. This malignant cancer is accompanied by early metastasis and has angiogenesis [11]. Invasive growth tumor, sometimes with no apparent bound areas with normal tissue, causes the removal of the tumor during surgery and results in annual mortality rates [12, 13]. Although recently, researchers tend to discover the genomic pathway of many oncogenic and repressive tumor pathways, proteomics studies indicate the magnitude of proteins in the tumor. Quantitative proteomics, as a useful biologic implement, allows the re-identification of the exact amount of proteins in biological samples [14]. In recent years, according to data from the expression of genes and proteins, researchers have investigated several proteins involved in the development of this pernicious cancer, which has been studied by tumor cells as distinct of clinical substitutes in malignant glioma [15]. Thus, the probable role of markers involved in the development and main cause of the spread of the tumor were examined in this study. One of the markers examined is a glial fibrillary acidic protein (GFAP), which is the main protein organized and almost individually in glial and astrocytes, member of medium-sized intermediate filament type III proteins and exist with the other type III intermediate proteins and some type IV-intermediate filaments in the cytoplasm [16]. It plays a fundamental role in maintaining the shape and motility of astrocytic processes and contributes to white matter architecture myelination.
Their structure is dynamic, active, and integral to muscle cells, the brain, and mesenchyme [16-18]. Another marker is Chitinase 3‐like 1 (YKL-40), a secreted glycoprotein with 40 KDa weight, located in an extracellular matrix, has multifunctional activity in aggressive cancers, and is considered as a chitinase-like protein. However, YKL-40 does not have catalytic activity compared to chitinases, which is due to the replacement of the amino acid in the binding site to the active site of the chitinases [19, 20].
The last factor is the insulin growth factor binding protein-2 (IGFBP-2). Among all the IGFBPs, IGFBP-2 has been involved in regulating insulin-like growth factor (IGF) activity in the nervous system, peripheral tissue, and organs [21]. The IGFBP-2 roles in regulating insulin activity are one of the growth factors of IGFs. Also, IGFBs inherently have independent features of IGFs [22]. Early studies demonstrated that IGFBP2 mRNA levels were maintained high during embryonic brain development but decreased in the adult brains, also directly or indirectly promotes transcriptional activation of a specific gene [23, 24]. Researchers have focused on several biomarkers based on information from the expression of genes and proteins during the disease [25]. In this regard, determination of serum survivin, hyaluronic acid, and laminin was used as potential tumor markers [26, 27]. The crosstalk between trace elements with DNA damage response, repair, and oxidative stress were reported in cancer [28].
Furthermore, some of the effective factors in disease pathogenesis and the interplay of klotho with signaling pathways in cancers were reported [29, 30]. Regulatory functions of circular RNAs and targeting the mammalian target of rapamycin signaling were reported in cancer [31, 32]. These markers are creating and spreading the tumor with a special role, which is probably due to the use of tumors from them. Besides evaluating these markers, they will clarify the biological status of the brain tumor and help detect the progression of this cancer. Given the devastating nature of GBM, biomarkers are required to assist in diagnosing, prognosis, and predicting treatment outcomes. This study aims to highlight the identity role of tissue and circulate GFAP, IGFBP-2, and YKL-40 for GBM as a combined profile with diagnostic and prognostic value.

Materials and Methods
Study population
A total of 22 patients with newly diagnosed GBM who had undergone surgery at the Erfan Hospital were included in the current study. Tumor size was calculated on preoperative  magnetic resonance imaging (MRI). All tumors had been examined histo-pathologically and classified according to the WHO classification. In this study, the levels of GFAP, YKL-40, IGFBP-2 were evaluated in 22 tumor tissues, and non-timorous matched adjacent tissue samples of patients with GBM using the enzyme-linked immunosorbent assay  (ELISA). Also, 22 healthy subjects with no history of GBM served as controls for plasma samples.
The inclusion criteria were patients who have been diagnosed with GBM. The exclusion criteria were patients who had a history of using anti-cancer drugs and patients with other malignancy. The patient’s written informed consent was obtained before collecting samples from all patients and healthy controls. The study has been approved by the Ethics Committee of Babol University of Medical Sciences (MUBABOL.HRI.REC.1396.197).
Tissue and plasma sample collection
For patients with GBM, blood samples had been taken immediately before surgery. Samples from volunteered subjects were collected in the absence of other illnesses.
Blood was drawn into tubes with the ethylenediaminetetraacetic acid anticoagulant.
The plasma was collected, and an analysis of protein levels by ELISA was done. For tissue samples, 2 ml of phosphate buffered saline (pH = 7.4) were added, and the samples were homogenized thoroughly by a homogenizer. Samples were then centrifuged at 3000 ×g for 20 min. collected the supernatants carefully.
Plasma and tissue GFAP, IGFBP-2, and YKL-40 measurements
Tumor and plasma samples were analyzed for GFAP, IGFBP-2, and YKL-40 using commercially available ELISA kits (Bioassay, China). All samples were tested in duplicate according to the manufacturer’s instructions. Plasma samples were diluted 1:10, and tissue samples were diluted 1:100. All standards and test specimens were run in with the volume of 40 µl pipette into each ELISA well. A biotin-labeled anti-GFAP-antibody, anti-YKL-40, and anti-IGFBP-2, specifically recognizing them, was employed as detector antibodies. A reading plate measured the absorbance at 450 nm. Total protein concentrations were determined as absorbance using a spectrophotometer at 450 nanometers (nm). The whole process of this study is demonstrated in Figure 1.
Statistical analysis
All analyses were evaluated using the SPSS version 22.0. The data were reported as the mean ± SD. Students’ t-test results were used for all comparisons. The p-value < 0.05 was considered statistically significant. Also, the ROC curve was used to check the diagnostic value of GFAP, YKL-40, and IGFBP-2.

Results
The result showed that the mean levels of GFAP, YKL-40, and IGFBP-2 in tissue samples of timorous of the patients with GBM were higher (0.61 ± 0.09,1.84 ± 0.73 and 48.01 ± 13.8, µg/ml respectively) when compared with non-tumorous brain tissue (0.30 ± 0.08, 0.86 ± 0.35, and 29.47 ± 13.80, µg/ml respectively). Nevertheless, the results showed that the mean levels of GFAP, YKL-40, and IGFBP-2 in plasma samples of the patients with GBM were lower (0.35 ± 0.32, 1.18 ± 2.08, and 2.30 ± 1.47, µg/ml respectively) when compared to healthy control (0.34 ± 0.31, 2.70 ± 6.25, and 23.24 ± 20.58, µg/ml respectively). Characteristics of the study population were demonstrated in Table 1. Clinical-pathological characteristics of the patient group were shown in Table 2. Levels of GFAP, IGFBP-2, and YKL-40 in tissue samples of tumorous and non-tumorous brain tissue of the patients with GBM were also indicated. As indicated in Table 3, levels of GFAP, IGFBP-2, and YKL-40 in tissue samples of tumors were markedly higher when compared with non-tumorous brain tissue of the patients with GBM (p=0.001).
However, the plasma levels of GFAP, IGFBP-2, and YKL-40 were not significantly different between the two groups (Table 4). Table 5 demonstrated the correlation between tissue levels of GFAP, IGFBP-2, and YKL-40. The diagnostic value of GFAP, IGFBP-2, and YKL-40 for differentiation of patients with GMB in tissue samples was also shown (Table 6). The diagnostic value of GFAP, IGFBP-2, and YKL-40 for the differentiation of patients with GBM in plasma samples was shown in Table 7. ROC curve for differentiating patients with GBM from healthy subjects in a tissue sample (Fig. 2). ROC curve for determining patients with GBM from healthy subjects in a tissue sample. ROC curve for determining patients with GBM from healthy subjects in a plasma sample (Fig. 3). Furthermore, ROC analysis in calculating the diagnostic accuracy for an optimized specificity obtained the cut-off point of 0.47, 1.24, and 37.44 µg/mg for GFAP, YKL-40, and IGFBp-2, respectively.

The gold standard method for GBM detection is neuro-imaging, but there are limitations to use this method, such as its high cost. Therefore, having the potential of markers for early detection is still the primary goal. This information stimulates us to find markers responsive to the earlier stages of cancer without any difficulty in detecting sera. In this regard, to overcome these shortcomings, circulating biochemical markers might be associated with GBM and could be evaluated as potential diagnostic biomarkers in patients with GBM since most current biomarkers cannot reach a certain level of sensitivities or specificities. Therefore, in the current study, GFAP, IGFBP-2, and YKL-40, which can be sampled non-invasively and cost-effectively, were evaluated as ideal biomarkers for patients with GBM.
The most important finding of the current study was a difference in GFAP level between tissue samples of tumorous and non-tumorous brain tissue of the patients with GBM. As expected, due to necrosis and the spread of brain cancer, the level of GFAP increased in patients, which is consistent with the results reported by researchers. It reported a significantly higher rate of this factor in brain tissue samples of patients with GBM [22]. Furthermore, ROC curve analysis confirmed that tissue GFAP acts as discriminatory to differentiate patients with GBM from non-tumorous brain tissue. According to the results of this study, the analysis of the ROC curve, the sensitivity, and specify of this biomarker was determined as 90% and 95% , respectively.
Contrary to our research, some researchers study on the brain tissue sample reported that the expression of this factor in the gradient IV glioma is reduced [33]. The results of this study showed that GFAP in plasma was unrelated to the study of researchers who reported GFAP as markers below 0.05 μg/L with 76% sensitivity and 100% specificity. It should be noted that in this study, the sample size of patients with GBM cancer was 50 [34]. One of the reasons for this research’s difference from other research is its small sample size and the different behavior of this marker in plasma and the tissue. Other researchers compared GFAP serum levels between various gliomas and non-glioma cases [35]. Also, the relationship between tumor size and GFAP was not found in this study, which was consistent with other studies [36].
Another finding of the present study was the existence of a difference in the YKL-40 between tissue samples of timorous and non-tumorous brain tissue of the patients with GBM. Researchers expected it to increase, which is in line with previous studies.
Other studies have shown that YKL40 -40 is an influential factor in angiogenesis [37, 38]. The ROC curve was analyzed, and the sensitivity and specificity of this biomarker were estimated at 81%, 81%, and the area under the curve was 0.916. Therefore, the YKL40 is suggested as an appropriate marker for tissue samples in patients with GBM. Nevertheless, there was no significant difference between the plasma YKL-40 levels in the patients with GBM compared to the healthy control group. This research suggested increased plasma YKL-40 values in patients with GBM patients [15]. Various effective factors in this area can be mentioned for these dereferences, including sample size, racial group, clinical and individual characteristics, and analysis methods or the technical platform.
The present study’s finding demonstrated a difference in the IGFBP-2 level between tissue samples of tumorous and non-tumorous brain tissue of the patients with GBM. Our results were consistent with the study by other researchers, which showed an increase of IGFBP-2 in a fourth grade glioma tissue specimen compared to healthy control [39]. Besides, by analyzing the ROC curve, the sensitivity and specificity of this biomarker in our study were 81% and 85%, respectively, and area under the curve was 0.85. Our results demonstrated that this marker is an appropriate marker for tissue samples in patients with GBM. In the present study, any significant correlation between tumor volume and IGFBP-2 level was not observed. A study conducted
by other researchers showed a significant correlation between serum IGFBP and tumor size [22]. Some researchers reported a correlation between IGFBP-2 and tumor volume and GBM development [40]. Also, in the present study, there was no significant correlation between plasma IGFBP-2 between two groups of patients with GBM and healthy control subjects, which contrasted with the study by other researchers, who had found a significant increase in IGFBP-2 factor in patients with GBM [39]. These differences can be explained by different factors, such as sample size, racial group, clinical and individual characteristics, and analysis methods or the technical platform. More importantly, a combined three-marker panel consisting of GFAP, IGFBP-2, and YKL-40 had a higher discriminatory power. Therefore, multiple panel-based markers could overcome shortcomings and provide better judgment. Further studies on larger groups of patients are required to confirm the results obtained.

Conclusion
In the current study, the tumor levels of all three tested proteins were significantly higher in patients with GBM compared with healthy controls, and all three tested proteins allowed for the differentiation of patients with GBM from the healthy controls. These results suggest that a combined profile of GFAP, IGFBP-2, and YKL-40 tissue levels might constitute a diagnostic approach to patients with GBM. Nevertheless, no difference has been found between the plasma levels of all three tested proteins in patients with GBM  compared with healthy controls. Future studies should investigate whether plasma gap, IGFBP-2, and YKL-40 could also be used to detect the GBM. We hope that our data will provide some insights into the detection of GBM.

Conflict of Interest
There are no competing interests to be disclosed.

Acknowledgments
The authors would like to thank Erfan Hospital surgery department staffs and Shirin Haghighifashi and Shahryar Pazhav for their assistance. The Babol University of Medical Sciences supported this study.

 
 
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