Tim-3 protein expression with MGMT methylation status in glioblastoma and association with survival

Background Understanding the molecular landscape of glioblastoma (GBM) is increasingly crucial for its therapy. Immune checkpoint molecules motivated the emergence of immune checkpoint-targeting therapeutic strategies. However, the prognostic significance of the immune checkpoint molecule T cell immunoglobulin mucin-3 (Tim-3) on tumor-infiltrating immune cells (TIICs) and O-6-Methylguanine-DNA methyltransferase (MGMT) methylation status remains to be fully elucidated. We aimed to develop an MGMT methylation status-associated immune prognostic signature for predicting prognosis in GBMs. Patients and Methods: A total of 84 patients with newly diagnosed GBM were involved. MGMT methylation status was retrospectively analyzed and the expression level of Tim-3 protein was investigated using immunohistochemistry (IHC). The correlation between Tim-3 protein expression and MGMT methylation status, and the prognosis was explored. Results The obtained data showed that Tim-3 protein was expressed at different levels in GBMs. Mesenchymal expression of Tim-3 protein in these tissues was 73.81% (62/84), including low 15.48% (13/84), moderate 7.14% (6/84) and strong expression 51.19% (43/84), respectively. Of the 48 patients whose tumors tested positive for MGMT methylation, the remaining 36 patients was negative. Conclusions We profiled the immune status in GBM with MGMT promoter methylation and established a local immune signature for GBM, which could independently identify patients with a favorable prognosis, indicating the relationship between prognosis and immune. MGMT promoter methylation with lower Tim-3 protein expression was statistically significantly associated with better survival. curve

approaches urgently requires a deeper understanding of the tumor's nature [3]. In addition to standard treatment involving surgery, radiotherapy and chemotherapy in GBM, immunotherapy is rapidly identified as a promising treatment modality [4]. A number of immune-related parameters have been reported for predicting the outcomes of patients with GBM [5,6]. MGMT promoter methylation status is significantly related to the prognosis of GBMs [7]. However, there is still a lack of studies which have systematically explored the MGMT promoter methylation status on immune microenvironment and their relationship with prognosis.
Tim-3 was widely expressed on mature T lymphocytes and macrophages [8]. Of note, with the exception of the immune response, increasing evidence has displayed that Tim-3 has functional roles in tumor biology [9]. Literatures suggests that Tim-3 is a promising target in glioma. But until now, no evidence has revealed the value of Tim-3 as a prognostic biomarker in GBM patients. The present study aimed to investigate the influence of MGMT promoter methylation on the immune microenvironment and develop an MGMT-associated immune prognostic signature for predicting prognosis in GBMs.

Materials And Methods Patients and specimens
A cohort of patients with histologically newly diagnosed GBM (WHO grade IV) was consecutively studied from July 2016 to January 2018. We only selected patients for whom affirmatory MGMT promoter methylation status, treatment course, and survival outcomes were known. Patients with a mixed history of cancer other than GBM and previous adjuvant radiotherapy or chemotherapy were excluded. Patients who died of diseases unrelated to gliomas were also eliminated from the study. The age ranged from 18 to 70 years old at the time of diagnosis. A series of 84 eligible patients who had tumor tissue available for testing were involved in this study. These patients received standard subsequent therapy by the Stupp protocol [10]. Follow up was regularly carried out. The overall survival (OS) was defined as the interval from GBM diagnosis until death or the last known followup for those who were censored.
Subsequently, the slides were washed in PBS and stained with 3, 3-diaminobenzidine (DAB). Finally, the slides were counterstained, dehydrated and mounted.

IHC assessment
The IHC number of Tim-3 protein expression was independently reviewed by two neuropathologists.

MGMT promoter methylation
We collected only patients who had documented MGMT promoter methylation testing of their initially resected tumor tissues. MGMT promoter methylation was confirmed by methylation-specific real-time PCR, according to our institutional practice.

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 7.0.0. The correlation between Tim-3 protein expression intensity with MGMT promoter methylation status and their prognosis was calculated with the chi-square test. Spearman's correlation analysis and statistical significance were used to evaluate correlations with gene expression. Independent prognostic factors for OS were identified using the Cox's proportional hazards model. OS curve was plotted with Kaplan-Meier method and the log-rank test was employed to assess the resulting survival curves. A probability value of less than 0.05 (p < 0.05) was regarded as significant.

Clinicopathological characteristics
Archival tissue samples from 84 patients with GBMs were enrolled in the study. Of the 84 GBMs patients, 43 were male (51.19%) and 41 female (49.81%). The median age at diagnosis was 41 years (range 18-70 years) and the median Karnofsky Performance Status (KPS) was 90 (range 70-100). All specimens were obtained from supratentorial area, identified by preoperative MRI. The follow-up duration ranged from 4 to 47 months and the median OS was 17.3 months.

Expression of Tim-3 protein
As the expression status of specific inhibitory receptors, Tim-3, programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and lymphocyte-activation gene 3 (LAG3), are associated with T cell exhaustion and immune escape, the Oncomine database was used to investigate the mRNA levels of these molecules in GBMs and normal tissues (Fig. 1c). Figure 1a showed that Tim-3 expression was remarkably high in GBMs than the corresponding normal tissues.
To validate the relationship of immune checkpoint molecules between Tim-3 and LAG3 as well as PD-1, the RNA-seq data in the TCGA database were exploited. As depicted in Fig. 1b, with a p-value threshold of 0.05, Tim-3 expression was significantly related with LAG3 and PD-1 in GBM. Tim-3 and other exhausting immune molecules (LAG3, PD-1, CTLA4, CD244, PD-1) exhibited significantly differential expression profiles between normal brain tissues and GBM tissues in TCGA database (https://cancergenome.nih.gov/), indicating their potential correlation with glioma progression (Fig. 1).
According to the immunohistochemical analysis, mesenchymal expression of immune checkpoint Tim-  Table 1). Notably, the strong expression of Tim-3 protein was more frequently observed in GBMs.

MGMT methylation status in 84 GBMs
Among the 84 patients, MGMT methylation status was analysed respectively. All patients had MGMT promoter methylation testing results. Testing was performed via methylation specific real-time PCR at the time of diagnosis. 48 patients whose tumors tested positive for MGMT methylation were determined to be MGMT methylated (57.14%) and 36 patients whose tumors tested negative for MGMT promoter methylation were MGMT unmethylated (42.76%) (Fig. 3, Table 1).

Correlation between MGMT methylation status with Tim-3 protein expression and survival
The relationship between the expressions of Tim-3 protein and the MGMT promoter methylation status are shown in Table 1

Discussion
Glioblastoma is the most common and lethal primary brain tumor, with strong risk of recurrence and short survival periods, and the cure of this formidable disease seems impossible [2,11]. Recent developments emphasize targeting the molecular characteristics and various approaches of immunotherapy. Although many new molecular markers have been identified, the MGMT promoter methylation status is commonly used in GBM studies [12].
The discovery of MGMT promoter methylation in GBMs as compared to its counterpart MGMT promoter nonmethylation, plays a key role in the understanding of glioma biology [13]. Increasing research displayed that the immunological tumor microenvironment of the gliomas differs based on their molecular property [14]. However, the mechanism controlling the association of MGMT promoter methylation status with the immune microenvironment is yet to be studied. The current study The present study is the first, to the best of our knowledge, to report that Tim-3 protein expression with MGMT promoter methylation status is critical in prognosis for patients with GBM. Tim-3 is an immune regulatory molecule, which motivates downstream cascade events upon stimulation by its ligand [9,15]. Emerging research has demonstrated the importance of Tim-3 in human tumorigenesis.
However, no further investigations have been made with respect to the role of Tim-3 protein expression with MGMT promoter methylation status in GBM patient prognosis. Aberrant expression of Tim-3 protein has been noticed to boost tumor progression and be associated with unfavorable prognosis in many kinds of caners [9,[16][17][18][19].
Tim-3 has previously been recorded to be expressed at strong levels in prostate cancer, hepatocellular carcinoma and melanoma [9,[16][17][18][19]. The protein level of Tim-3 in the GBM tissues were initially examined using immunohistochemical analyses. We observed the expression of Tim-3 protein in the GBM interstitial tissue. In line with previous reports, the present study found that the expression of Tim-3 protein was significantly stronger in GBM samples without MGMT methylation.
Tim-3 can efficiently predict the aggressive behavior of head and neck squamous cell carcinomas [20]. In prostate cancer, Tim-3 overexpression results in the attenuated level of tumor suppressor FLRT3 and increased expression of genes such as MMPs to trigger invasion and metastasis [21]. Tim-3 promotes glioma cell proliferation and increased level of Tim-3 enhances angiogenesis by inducing transdifferentiation of glioma stem cells into endothelial cells and stabilization of vascular base membranes, which is implicated as a mechanism that Tim-3 furtherances the progression of gliomas [22]. But in that study, the authors did not explore the association between Tim-3 protein level and the prognosis of glioma patients, probably due to the rare number of glioma specimens. Therefore, the prognostic significance of Tim-3 in gliomas remains unclear.
Molecular genetic testing, in particular MGMT promoter methylation, is nowadays considered as a predictive factor for standard chemotherapy in GBM [23]. In the study, 57.14% of the responders has a routine determination of MGMT promoter methylation. The study concluded methylated patients had better results with temozolomide [24,25]. A single center study observed that MGMT promoter methylation is an independent prognostic factor associated with a good outcome, including prolonged progression-free survival (PFS) and OS in GBM [26]. In order to look into this relationship between MGMT promoter methylation status and the expression of Tim-3 protein in GBMs, we retrieved 84 specimens from the tumor tissue bank. In line with previous findings, Tim-3 protein was expressed in different level in GBM tissues. High Tim-3 protein expression was more frequently found in GBMs without MGMT promoter methylated patients, which was capable of predicting poor prognosis of GBM patients. Among these 84 patients, the median OS was 17.3 months. The survival time for those GBM patients with low Tim-3 protein expression with MGMT promoter methylation was longer than those with mediate and strong Tim-3 expression. Subsequent multivariate cox regression analyses confirmed that Tim-3 protein expression with MGMT promoter methylation was an independent prognostic factor for GBM patients. The subgroup analysis revealed that strong Tim-3 protein expression with MGMT promoter non-methylation was remarkably correlated with the short survival for patients. The average survival time, however, hardly differs in those patients with unexpressed and mediate Tim-3 expression.
We also observed that high-risk GBM patients had higher levels of Tim-3 protein with unmethylated MGMT. An MGMT-associated immune prognostic signature, which was related to prognosis, was constructed. In addition, the immune prognostic signature provides novel insights into the GBM immune microenvironment and potential immunotherapies, which enabled us to classify patients into   Immunohistochemical staining of Tim-3 protein expression in the formalin-fixed, paraffinembedded GBM tissues.

Figure 4
Immunohistochemical staining of Tim-3 protein expression in the formalin-fixed, paraffinembedded GBM tissues.

Figure 5
The bar graph shows methylated and unmethylated MGMT distribution in different Tim-3 protein expression group.

Figure 6
The bar graph shows methylated and unmethylated MGMT distribution in different Tim-3 protein expression group.

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