Original Article Over-Expression of TOP2A As a Prognostic Biomarker in Patients with Glioma

Int J Clin Exp Pathol 2018;11(3):1228-1237 www.ijcep.com /ISSN:1936-2625/IJCEP0070536

Original Article Over-expression of TOP2A as a prognostic biomarker in patients with glioma

Tianmin Zhou1, Yan Wang2, Dongmeng Qian1, Qing Liang1, Bin Wang1

1Key Laboratory of Medicine and Biotechnology of Qingdao, Department of Microbiology, Medical College of Qingdao University, Qingdao, Shandong, P. R. China; 2Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P. R. China Received December 7, 2017; Accepted January 19, 2018; Epub March 1, 2018; Published March 15, 2018

Abstract: Topoisomerase (DNA) II alpha (TOP2A), an enzyme that controls and alters the topologic states of DNA during transcription, is aberrantly expressed in many cancers. However, few studies have investigated expression of TOP2A and its clinical significance in glioma. We retrieved six independent investigations from the Oncomine database and found that TOP2A is highly expressed in glioma tissues compared with corresponding normal controls. Similar results were also found in clinical specimens at the protein level. Immunohistochemical analysis indicated that TOP2A over expression was highly correlated with grade stage, KI67 positive percentage, IDH1 mutation, and age, but other clinical parameters such as sex distribution and tumor size were barely associated with high TOP2A gene expression. Meanwhile we used Prognos can to assess the prognostic value of TOP2A expression in glioma patients, and found that high expression was associated with poor prognosis of patients with glioma. Furthermore, we used the Gene-Cloud of Biotechnology Information (GCBI) bioinformatics platform predict the role of TOP2A in glioma. It was not only involved in DNA replication, chromosome condensation, and responses to DNA damage stimuli, but also promoted cancer cell mitotic cell cycle and apoptosis, and phosphatidylinositol-mediated signaling by regulating gene expression. By these approaches we demonstrate that TOP2A may be a reliable prognostic factor or therapeutic target in glioma.

Keywords: TOP2A, glioma, marker, prognosis

Introduction DNA during transcription. This nuclear enzyme is involved in processes such as chromosome Glioma remains the most common adult brain condensation, chromatid separation, and the tumor which is slightly more common in men relief of torsional stress that occurs during than in women [1], with 7.3 cases pr. 100,000 DNA transcription and replication [6]. Its aber- person-years. High-grade gliomas are present rant expression has been associated with mu- in 85% and low-grade glioma in 15% with 5- ltiple tumor prognostics [7], including lung year overall survival of 82, 54, 22 and 3% for cancer, breast cancer, pancreatic adenocarci- grade I, II, III and IV, respectively [2]. Within a noma, and colorectal cancer [8-11]. There five year follow-up time, most patients had a are few investigations analyzing expression of recurrent tumor and died of the disease [3]. TOP2A in glioma [12], and due to the limited number of studies and few clinical samples, no Despite much effort focused on improving the role has been detected. diagnosis and therapy of glioma, the clinical outcome of glioma is still unsatisfactory [4, In this study, we extended glioma research uti- 5]. Consequently, more studies are needed to lizing large databases and clinical samples, explore the potential mechanism and detect with the purpose of determining the expressi- effective diagnostic and prognostic biomark- on level of TOP2A in glioma and its corres- ers to prolong the lives of glioma patients. ponding prognostic value. Using the Gene- Cloud of Biotechnology Information (GCBI) bio- TOP2A gene encodes a DNA topoisomerase informatics platform we investigated the role that controls and alters the topologic state of of TOP2A of glioma. Over-expression of TOP2A gene in patients with glioma

Materials and methods sue specimens from these patients, permis- sion from the Affiliated Hospital of Qingdao Oncomine analysis University was granted. (5) A representative sample was taken from each FFPE tissue and The Oncomine database (http://www.oncomi- hematoxylin and eosin (HE) staining was per- ne.org) incorporates 264 independent datas- formed. Results were reviewed by two experi- ets including 35 cancer types and supports enced pathologists. various methods of analysis, including molecular concepts, interactome, and meta-analysis Immunohistochemical staining and evaluation [13]. Thus, we detected the level of TOP2A in different cancers including hematological A traditional immunohistochemical (IHC) stain- malignancies and solid tumors and adopted ing protocol was used in this study. Brieﬂy, tis- the Oncomine database online tools meta-an- sue slices was deparaffinized in xylene and alysis to estimate the expression of TOP2A in rehydrated with different concentrations of et- glioma and normal brain control that come hanol alcohol. Then the section was treated from 6 different datasets. with 3% hydrogen peroxide, followed by anti- gen retrieval with 10 mM citrate buffer (pH PrognoScan 6.0) with microwave. After being blocked with 10% goat serum for 30 min, incubation with PrognoScan (http://www.prognoscan.org) is a the primary antibody was done overnight at comprehensive platform for evaluating pot- 4°C with anti-TOP2A antibody (mouse onoclo- ential tumor biomarkers and therapeutic tar- nal antibody, 1:100 dilution, ZM-0245, Zhong- gets. Based on a large collection of cancer shanjinqiao, China) overnight at 4°C, followed microarray datasets with clinical annotation, by a peroxidase-labeled secondary antibody. PrognoScan is a useful online tool for assess- Immunoreaction score distribution (IOD) mea- ing the association between specific gene ex- surement, as reported previously, was used pression and prognosis in patients with can- to determine the positive-staining density [16]. cer [14]. We used the PrognoScan platform to A Leica CCD camera DFC420 connected to a validate the prognostic value of TOP2A expr- Leica DM IRE2 microscope (Leica Microsyst- ession in patients with glioma. ems Imaging Solutions, Ltd., Cambridge, UK) was used as an imaging system. High-power Patients and specimens magnification (×40) was used to photograph representative fields. The IODs in each image Forty-six glioma tumor tissue samples were were measured and counted using Image-Pro collected from the Affiliated Hospital of Qing- Plus v6.0 software (Media Cybernetics, Inc., dao University at Qingdao in China from July Bethesda, MD, USA). 2014 to December 2016. All the patients were with tumors suitable for resection accord- X-tile analysis ing to the Chinese guideline of surgical treatment strategy for glioma in 2012, and among TOP2A expression was assessed with X-tile them, 16 cases were in gradeII, 9 cases were plots. TOP2A expression was expressed as grade III, and 21 cases were glioma. Altoge- IOD and the optimization of cutoff points was ther, 46 cases of formalin-fixed paraffin-em- based on outcomes. The cutoff score derived bedded (FFPE) specimens were used for Im- from 46 cases of training set by a standard munohistochemistry. log-rank method was used to assess statistical significance. P values were obtained from In this study, the inclusion criteria were as a lookup table. the following: (1) Pathological diagnosis of glioma tumors; (2) A diagnosis that was cons- Gene-cloud of biotechnology information istent with the histological diagnostic criteria (GCBI) of the World Health Organization [15]; (3) Tu- mor is the primary tumor in the brain, and pa- GCBI (Shanghai, China, https://www.gcbi.com. tients did not receive pre-operative anti-can- cn) is a platform comprising a variety of re- cer treatment, and there were no extrahepa- search findings, genetic information, sample tic metastases; (4) For the use of archived tis- information, data algorithms and bioinformat-

1229 Int J Clin Exp Pathol 2018;11(3):1228-1237 Over-expression of TOP2A gene in patients with glioma

Figure 1. Analysis of TOP2A expression by Oncomineonline database. A: The mRNA expression of TOP family mem- bers in different tumor types. This graph- ic shows the numbers of datasets with statistically significant mRNA over-expression (red) or down-expression (blue) of the target gene (cancer vs. normal tissue). The P value threshold is 0.01. The number in each cell represents the number of analyses that meet the threshold within those analysis and cancer types. The gene rank was analyzed by percentile of target gene in the top of all genes measured in each research. Cell color is determined by the best gene rank percentile for the analyses within the cell. B: TOP2A expression is up-regulated in glioma. A meta-analysis of TO2PA gene expression from six Onco- mine databases where colored squares indicate the median rank for TOP2A (vs. Normal tissue) across 8 analyses. C: Comparison of TOP2A mRNA expression in TCGA dataset. D: Comparison of TOP2A mRNA expression in Sun L etal. dataset. Comparison the expression levels of TOP2A in paired glioma tumor and peri-tumoral tissues.

ics. It creates a “gene knowledge base” which encompasses biolo- gy, medicine, informatics, comput- er science, mathematics, graphics, and other disciplines. GCBI includ- es more than 120 million copies of genomic samples, approximately 90,000 copies of tumor samples and more than 17 million copies of genetic information. Therefore, GC- BI is a good bioinformatics analysis platform and has provided data analysis support for many studies on cancer research [17-20]. In this study, we used GCBI to identify di��f- ferentially expressed genes (DEGs) between glioma tissue and normal brain control and finally predict the role of TOP2A in gliomas.

Statistical analysis

All the statistical analyses were ca- rried out using SPSS 17.0 software (SPSS Inc, Chicago, IL, USA) and GraphPad Prism 5 (San Diego, CA) software. The relationship between TOP2A expression and clinicopathological factors was analyzed by Chi-squared test and Analysis of

1230 Int J Clin Exp Pathol 2018;11(3):1228-1237 Over-expression of TOP2A gene in patients with glioma

Table 1. Meta-analysis of TO2PA gene expression from six Oncomine databases Fold Overexpression Datasets (sample size) Comparison groups P-value Change Gene Rank Sun Brain (180) Glioblastoma vs. Normal 102.544 2.37E-27 22 (in top 1%) Oligodendroglioma vs. Normal 28.449 8.4E-18 27 (in top 1%) Anaplastic Astocytoma 34.640 4.10E-12 27 (in top 1%) Liang Brain (33) Glioblastoma vs. Normal 2.202 6.27E-6 115 (in top 2%) TCGA Brain (552) Glioblastoma vs. Normal 28.903 2.59E-13 115 (in top 2%) French Brain Statistics (29) Anaplastic Oligodendroglioma VS. Brain 9.685 1.15E-8 115 (in top 2%) Shai Brain Statistics (34) Glioblastoma vs. White Matter 2.291 1.17E-6 227 (in top 3%) Murat Brain Statistics (84) Glioblastoma vs. Normal 13.755 2.17E-11 262 (in top 2%) Note: A meta-analysis of TO2PA gene expression from six Oncomine databases where colored squares indicate the median rank for TOP2A (vs. Normal tissue) across 8 analyses. French Brain (1), Liang Brain (2), Murat Brain (3), Shai Brain (4), Sun Brain (5-7) and TCGA (8). The P value is given for the medianrank analysis.

Figure 2. TOP2A expression levels in peri-tumoral tissues (A) and glioma (B) (×40). (C) Box plot indicating the mean staining intensity of paired glioma and peri- tumoral tissues. (D) Paired peri-tumoral tissue and glioma tissue in the same section (D) (×10).

Variance. Survival curves for TOP2A expression were generated using the Kaplan-Meier method and compared using the log-rank test. P values < 0.05 were considered statistically significant. Each statistical analysis of online database was completed by usedits online tool.

Results

TOP2A is distinctively overexpressed in glioma than normal brain

Firstly we used Oncomine database to analysis the expression of TOP2A in human cancers, including hematological malignancies and solid tumors (Figure 1A). We found that TOP- 2A mRNA expression was high in different cancer types than corresponding normal samples across a wide variety of datasets. Then in order to assess the expression of TOP2A in glioma, we analyzed six independent microarray datasets from Oncomine database [21- 26], and revealed statistically significant over expression of TOP2A in the majority of glioma tissues compared with normal brain controls (Table 1). The median rank of TOP2A in up regulated genes of TOP2A was 126.0 based on a meta-analysis across the six datasets, including 8 analyzed using Oncomine algorithms [27] (P = 3.14*10-6, Figure 1B) (In total, 859 glioma samples and 53 healthy controls were used). TOP2A transcripts were 28.90 fold elevated in glioma samples as compared with

1231 Int J Clin Exp Pathol 2018;11(3):1228-1237 Over-expression of TOP2A gene in patients with glioma

samples as compared with normal tissues (P = 2.37*10- 27) (Figure 1D).

Elevation of TOP2A in glioma compared with peri-tumoral tissues by immunochemistry

We also analyzed the expression levels of TOP2A protein in 46 glioma tumors and paired peri-tumoral tissues by immunochemistry (IHC). Expression of TOP2A protein levels was frequently higher in the glioma tumor tissues (Figure 2A) than in peri-tumoral tissues (Figu- re 2B). These differences were statistically significant Fi- ( gure 2C; P < 0.0001, paired t test). Meanwhile we found that expression of TOP2A is positive in all samples of glioma. In 43 cases, glioma tissues and peri-tumoral tissue were located in one area, and the picture clearly revealed that TOP2A expression was higher in peri-tumoral tissue than glioma tissue (Figure 2D).

Figure 3. Correlation analysis of TOP2A with clinicopathological features of Association of TOP2A expres- glioma. A, B: Expression of TOP2A was significantly associated with grade stage. C: KI67 positive percentage. sion with clinicopathological features of glioma patients

Table 2. Association of TOP2A expression with As show in Figure 3A and 3B, Immunoreaction pathological characteristics of glioma (46 score distribution (IOD) of TOP2A significant- cases) ly increased along with grad stage of glioma (F Pathological Case TOP2A = 11.91, P < 0.0001) and KI67 positive per- P-value category number Low High centage (Spearman’s r = 0.7788, P < 0.0001). We also learned the association of the expr- Sex Male 29 11 18 0.1264 ession of TOP2A with other clinicopathological Female 17 11 6 features in glioma, as shown in Table 2. Ex- Age ≥ 50 23 5 18 0.0009 pression of TOP2A association correlated wi- < 50 23 17 16 th IDH1 mutation (P = 0.0113) and age (P = Tumor size ≥ 3 cm 21 10 11 1.0000 0.0009), but other clinical parameters, such as < 3 cm 25 12 13 sex distribution (P = 0.1264) and tumor size (P IDH1 IDH1 (+) 15 12 3 0.0113 = 1.0000) were barely associated with high IDH1 (-) 16 5 11 TOP2A gene expression.

High mRNA expression of TOP2A is an unfavor- normal tissues in a dataset with 552 samples able prognostic factor for glioma that derived from TCGA database [21] (Figure 1C). In another dataset from Sun L study [22], Because many of the above data show that TOP2A was 102.544 fold elevated in glioma TOP2A is highly expressed in gliomas. Ac-

1232 Int J Clin Exp Pathol 2018;11(3):1228-1237 Over-expression of TOP2A gene in patients with glioma

2925 (P < 0.01, Flod change > 2) significant DEGs were identified, of which 1041 over-pre- sented and 1884 showed an attenuated be- havior (Figure 5B and 5C). We found that TOP- 2A was significantly overexpressed in glioma samples (Fold Change = 6.447, t = 17.9485, P < 0.01). We then performed a Gene Ontology analysis of 2925 differentially expressed genes (Figure 5D). Fortunately, we found that TOP2A in the enriched set was associated with mitotic cell cycle, positive regulation of transcription from RNA polymerase II promoter, positive regulation of apoptotic process, DNA replication, phosphatidylinositol-mediated signaling, response to DNA damage stimulus, chromosome condensation. So we speculate that the elevation of TOP2A not only takes part in DNA replication, chromosome condensation, and response to DNA damage stimulus, but also associates with mitotic cell cycle, apoptotic processes, and phosphatidylinositol- Figure 4. Kaplan-Meier survival curves generated mediated signaling. from PrognoScan for TOP2A mRNA expression in patients with glioma. A: Overall survival curves for pa- Discussion tients with glioma in the GEO dataset GSE4271. B: Patients with glioma in the GEO dataset GSE4412. Glioma, a tumor of central nervous system, HR = hazard ratio. which mainly develops from the macroglial cells, presents the highest prevalence and mo- cordingly, we hypothesized that TOP2A expr- rtality risk. The difficult treatment of patients ession may be a prognostic factor for patients with glioma is primarily attributed to recurren- with glioma. We used the PrognoScan data- ce and resistance to chemoradiotherapy [29- base to analyze the correlation of TOP2A ex- 31]. Despite the challenges of treatment, it is pression with overall survival in GSE4271 and rewarding to illustrate the pathogenesis of gli- GSE4412 enrolled from the Gene Expression oma, as well as to develop novel prognostic Omnibus (GEO) datasets with a total of 153 strategies and discover effective therapeutic cases. The results show that TOP2A expres- approaches. TOP2A is a nuclear enzyme that sion is highly associated with overall survival mainly involved in processes such as chro- of glioma patients (log-rank test, P < 0.001, mosome condensation, chromatid separation, P = 0.0016, Figure 4A and 4B). and the relief of torsional stress that occurs during DNA transcription and replication [6]. It Prediction of the role of TOP2A in glioma has been reported that TOP2A is a sensitive and specific marker in actively proliferating The elevated expression of TOP2A suggesting cells (in the late S, G2, and M phases of the cell that it they might play unique roles in the de- cycle) which suggests a role in a wide range velopment of glioma, We used Gene Expres- of human cancers [32, 33]. sion Omnibus (GEO) datasets GSE4290 which consists of 157 glioblastoma samples and 23 In the present study, through the Oncomine non-neoplastic epilepsy samples on the Gene- databases, we found that TOP2A was signifi- Cloud of Biotechnology Information (GCBI) bio- cantly over expressed in glioma compared with informatics platform to identify differentially normal brain samples. Similar results were expressed genes (DEGs) (seen in Figure 5A). found in 46 gliomas and paired peri-tumoral Following a standard protocol [28] of sample tissues by immunochemistry byanalysis of the qualification and normalization, raw expression association of TOP2A expression with clinico- values were summarized and analyzed in a pathological features of glioma patients. TOP- consecutive workfow based on GCBI. In total, 2A significantly increased along with grade

1233 Int J Clin Exp Pathol 2018;11(3):1228-1237 Over-expression of TOP2A gene in patients with glioma

Figure 5. GCBI database Predict the role of TOP2A in glioma. A: Flow dia- gram of the study design; B: Volcano plot for potential DEGs (n = 180) (glioma n = 157, in yellow; normal lung tissues n = 23, in blue); X-axis was log2 (Fold Change), Y-axis was -log10 (p value). C: Heat map for potential DEGs (n = 180) (glioma n = 157, in yellow; normal lung tissues n = 23, in blue); D: Gene ontology (GO) enrichment analysis the DEGs in glioma.

1234 Int J Clin Exp Pathol 2018;11(3):1228-1237 Over-expression of TOP2A gene in patients with glioma stage of glioma, KI67 positive percentage, R. China. E-mail: [email protected]; Bin Wang, IDH1 mutation and age, but other clinical School of Basic Medical Sciences, Qingdao Uni- parameters, such as sex distribution and tu- versity, Qingdao, P. R. China. Tel: +86-131-0772-05- mor size were barely associated with high 70; E-mail: [email protected] TOP2A gene expression. However, there was no evidence revealing arole of highly express- References ed TOP2A in the prognosis of glioma. In our [1] Cancer Genome Atlas Research Network, Brat study, according to the PrognoScan database, DJ, Verhaak RG, Aldape KD, Yung WK, Salama we could find its prognostic value: high expr- SR, Cooper LA, Rheinbay E, Miller CR, Vitucci ession of TOP2A caused shorter overall survi- M, Morozova O, Robertson AG, Noushmehr H, val time and shorter disease-free survival time. Laird PW, Cherniack AD, Akbani R, Huse JT, Thus, the expression of TOP2A might become Ciriello G, Poisson LM, Barnholtz-Sloan JS, a potential biomarker for the prognosis of Berger MS, Brennan C, Colen RR, Colman H, glioma. Flanders AE, Giannini C, Grifford M, Iavarone A, Jain R, Joseph I, Kim J, Kasaian K, Mikkelsen T, As for the functional and pathway enrichment Murray BA, O’Neill BP, Pachter L, Parsons DW, analysis, TOP2A was strongly emphasized in Sougnez C, Sulman EP, Vandenberg SR, Van cell cycle pathways and DNA replication pro- Meir EG, von Deimling A, Zhang H, Crain D, Lau cesses, which areconnected with its physiolo- K, Mallery D, Morris S, Paulauskis J, Penny R, gical functions [34, 35]. In our study, GCBI anal- Shelton T, Sherman M, Yena P, Black A, Bowen J, Dicostanzo K, Gastier-Foster J, Leraas KM, ysis found that mitotic cell cycle, positive regu- Lichtenberg TM, Pierson CR, Ramirez NC, Tay- lation of transcription from RNA polymerase II lor C, Weaver S, Wise L, Zmuda E, Davidsen T, promoter, positive regulation of apoptotic pro- Demchok JA, Eley G, Ferguson ML, Hutter CM, cess, DNA replication, phosphatidylinositol-me- Mills Shaw KR, Ozenberger BA, Sheth M, Sofia diated signaling, response to DNA damage HJ, Tarnuzzer R, Wang Z, Yang L, Zenklusen JC, stimulus, and chromosome condensation pa- Ayala B, Baboud J, Chudamani S, Jensen MA, thways were highly enriched in glioma sampl- Liu J, Pihl T, Raman R, Wan Y, Wu Y, Ally A, Au- es with TOP2A up-regulated. Furthermore, ear- man JT, Balasundaram M, Balu S, Baylin SB, lier clinical data indicate that TOP2A mRNA Beroukhim R, Bootwalla MS, Bowlby R, Bristow and protein expression might participate in the CA, Brooks D, Butterfield Y, Carlsen R, Carter S, Chin L, Chu A, Chuah E, Cibulskis K, Clarke A, process of chromosome condensation, chro- Coetzee SG, Dhalla N, Fennell T, Fisher S, Ga- matid separation, and the relief of torsional briel S, Getz G, Gibbs R, Guin R, Hadjipanayis stress that occurs during DNA transcription and A, Hayes DN, Hinoue T, Hoadley K, Holt RA, replication [6]. According to our analysis, we Hoyle AP, Jefferys SR, Jones S, Jones CD, speculate that high expression of TOP2A at Kucherlapati R, Lai PH, Lander E, Lee S, Lich- mRNA and protein levels might be one of the tenstein L, Ma Y, Maglinte DT, Mahadeshwar causes of cell cycle apoptotic process and HS, Marra MA, Mayo M, Meng S, Meyerson ML, phosphatidylinositol-mediated signaling. Mieczkowski PA, Moore RA, Mose LE, Mungall AJ, Pantazi A, Parfenov M, Park PJ, Parker JS, In conclusion, we used bioinformatics and Perou CM, Protopopov A, Ren X, Roach J, immunochemistry analysis to define the expr- Sabedot TS, Schein J, Schumacher SE, Seid- ession level of TOP2A in glioma. TOP2A was man JG, Seth S, Shen H, Simons JV, Sipahima- identified in association with the progression lani P, Soloway MG, Song X, Sun H, Tabak B, Tam A, Tan D, Tang J, Thiessen N, Triche T Jr, and prognosis of glioma, probably regulating Van Den Berg DJ, Veluvolu U, Waring S, Weisen- cell cycle apoptotic processes and phosphati- berger DJ, Wilkerson MD, Wong T, Wu J, Xi L, Xu dylinositol-mediated signaling. AW, Yang L, Zack TI, Zhang J, Aksoy BA, Arach- chi H, Benz C, Bernard B, Carlin D, Cho J, Di- Disclosure of conflict of interest Cara D, Frazer S, Fuller GN, Gao J, Gehlenborg N, Haussler D, Heiman DI, Iype L, Jacobsen A, None. Ju Z, Katzman S, Kim H, Knijnenburg T, Kreis- berg RB, Lawrence MS, Lee W, Leinonen K, Lin Address correspondence to: Tianmin Zhou, Key P, Ling S, Liu W, Liu Y, Liu Y, Lu Y, Mills G, Ng S, Laboratory of Medicine and Biotechnology of Qing- Noble MS, Paull E, Rao A, Reynolds S, Saksena dao, Department of Microbiology, Medical College G, Sanborn Z, Sander C, Schultz N, Senbabao- of Qingdao University, 308 Ningxia Road, Qingdao, P. glu Y, Shen R, Shmulevich I, Sinha R, Stuart J,

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