Hindawi
BioMed Research International
Volume 2018, Article ID 7945845, 12 pages
https://doi.org/10.1155/2018/7945845

Research Article
Elevated TERT Expression in TERT-Wildtype Adult
Diffuse Gliomas: Histological Evaluation with a Novel
TERT-Specific Antibody
Kenta Masui,1 Takashi Komori ,2 Yukinari Kato,3 Kenkichi Masutomi,4Koichi Ichimura,5 Satoshi Ogasawara,3 Mika K. Kaneko,3 Hiroharu Oki,3Hiroyoshi Suzuki,6 Masayuki Nitta,7 Takashi Maruyama,7 Yoshihiro Muragaki,7Takakazu Kawamata,7 Tatsuo Sawada,1 and Noriyuki Shibata1
1

Department of Pathology, Tokyo Women’s Medical University, Tokyo 162-8666, Japan
Department of Neuropathology, Tokyo Metropolitan Neurological Hospital, Tokyo 183-0042, Japan3
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi 980-8575, Japan
4
Division of Cancer Stem Cell, National Cancer Center Research Institute, Tokyo 104-0045, Japan
5
Division of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo 104-0045, Japan
6
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Miyagi 983-0045, Japan
7
Department of Neurosurgery, Tokyo Women’s Medical University, Tokyo 162-8666, Japan
2

Correspondence should be addressed to Takashi Komori; komori-tk@igakuken.or.jp

Academic Editor: Jens Schittenhelm
Copyright © 2018 Kenta Masui et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Telomerase reverse transcriptase (TERT) is important for the biology of diffuse gliomas. TERT promoter mutations are selectively
observed among 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene- (IDH-) wildtype glioblastoma (GBM).
However, TERT transcripts range widely in various cancers including gliomas, and TERT protein expression has been rarely
investigated thus far. It would be thus critical to examine the expression level of TERT in tumors in addition to its mutational
status, and sensitive and specific methods are urgently needed to examine TERT protein expression for the assessment of TERT
biology in gliomas. Using our newly developed TERT-specific monoclonal antibody (TMab-6) applicable to human tissue, we foundan unexpected increase in TERT expression in TERT-wildtype as well as TERT-mutated gliomas and in tumor vasculature. Thisis the first extensive analysis on the expression of TERT immunoreactivity in human glioma tissue, suggesting that TERT protein
expression may be regulated by several mechanisms in addition to its promoter mutation.

1. Introduction
Diffuse gliomas in adults are now separated into threecomprehensive tumor groups with distinctive prognoses,
based on the status of isocitrate dehydrogenase genes 1 and
2 (IDH1/2) mutations and 1p/19q-codeletion [1]. Recently,
hotspot mutations in the telomerase reverse transcriptase
gene (TERT) promoter (chromosome 5: 1,295,228 C>T and
1,295,250 C>T) have been identified in melanomas [2, 3]. The
subsequent investigation in a series of gliomas demonstrated
the applicability of the TERT promoter mutations in theclassification of diffuse gliomas [4], and the selectively highfrequency of the TERT promoter mutations was observed
among 1p/19q-codeleted oligodendrogliomas and IDHwildtype glioblastoma (GBM) [5]. The presence of TERT
promoter mutations is well-correlated with three distinctive
glioma groups [1], suggesting that TERT could be deeply
involved in the biology of diffuse gliomas.
Hotspot mutations in the TERT promoter (C228T and
C250T) are currently recognized as the only event in gliomas
to upregulate TERT transcripts, and mRNA of TERT tends
to be increased in TERT-mutated gliomas compared withnormal brain tissue or TERT-wildtype gliomas [2, 3, 6].
However, the TERT transcripts range widely in expression

2in various cancers including gliomas [5, 7], and there may
be other potential mechanisms to upregulate TERT mRNAin addition to its promoter mutations. Indeed, in other brain
tumors and systemic cancers, epigenetic shifts in the TERT
promoter regions including DNA methylation and histone
modifications were reported to regulate TERT transcripts
and reactivation [8–10], necessitating a rethinking of themechanism to upregulate the expression of TERT.
As an essential component of telomerase, TERT adds
telomeric repeats to chromosomal ends using telomerase
RNA component (TERC) as a template and maintains
telomere length. Telomerase activity had been considered tobe maintained in most types of the cancer cells, enablingthem to proliferate and survive indefinitely [11]. However, a
recent report demonstrated that telomere length is positively
correlated with mutations in the alpha thalassemia/mental
retardation syndrome X-linked gene (ATRX) but not the
TERT promoter, suggesting that TERT upregulation itself
may be essential for glioma biology irrespective of telomere
length [12]. It would be thus critical to examine the expression
level of TERT protein in diffuse gliomas to clarify the role of
TERT in their biology in addition to its mRNA level as wellas mutational status, but few reports have so far validated
the expression of TERT protein in human glioma tissue[7]. Sensitive and specific methods are strongly desired to
examine TERT protein expression for the future analyses on
the TERT biology in diffuse gliomas.
In this study, we developed a TERT-specific antibody
applicable to human tissue and examined the expression of
TERT in a series of glioma samples. Although the practical
diagnostic utility of the antibody to detect the TERT-mutatedtumors was expected, TERT immunohistochemistry was
not capable of identifying the differences between TERTmutated gliomas and TERT-nonmutant gliomas. Surprisingly, however, an increase in TERT protein expression
was detected across all types of gliomas including tumors
with wildtype TERT, and the level of TERT expression
was different even among the TERT-mutant gliomas suchas oligodendrogliomas and GBMs. Our study is the first
thorough analysis on the expression of TERT protein using a
specific TERT monoclonal antibody in human glioma tissue,
suggesting that TERT protein expression may be regulated byseveral mechanisms in addition to its promoter mutation.

2. Materials and Methods
2.1. Tissue Collection. A total of 41 human glioma tissues
and 4 nonneoplastic adult human cerebral tissues fromfour epileptic patients were obtained at surgery from Tokyo
Women’s Medical University Hospital and Tokyo Metropolitan Neurological Hospital. The tumor samples included 11
grade II and III astrocytic tumors with IDH mutations[diffuse astrocytoma (DA) and anaplastic astrocytoma (AA)],
10 grade II and III oligodendroglial tumors, 11 grade IV
IDH-wildtype GBMs, and 9 IDH-wildtype diffuse astrocytomas including tumors histologically diagnosed as diffuse
astrocytoma, anaplastic astrocytoma, oligoastrocytoma, andanaplastic oligoastrocytoma which did not show IDH mutations, p53 immunopositivity, and the loss of ATRX expression

BioMed Research International(wtIDH/p53-/ATRX+). Clinical information of the cases wascollected from the medical records. All specimens were
fixed in 10% buffered formalin and embedded into paraffin
sections. Sections were routinely stained with hematoxylin
and eosin (H&E), and histopathological diagnoses were madeaccording to the World Health Organization (WHO) 2016classification of tumors of the nervous system [13–15] andInternational Society of Neuropathology-Haarlem consensus
guidelines [16].
All tumor and control brain samples were subjected tohistological diagnosis followed by molecular genetics screening for a more precise/molecular diagnosis (Figure 1(a)) [17].
Tumors in the astrocytoma group (DA/AA) were characterized by detectable immunoreactivity for mutated IDH1
(R132H) and p53 [18] and the loss of immunoreactivity for
ATRX [19, 20]. All tumors in the oligodendroglioma groupdemonstrated mutated IDH1 (R132H) and 1p/19q-codeletion.
GBM tumors did not possess mutations in IDH, corresponding to “GBM, IDH-wildtype” [14, 21]. The tumors in the
IDH-wildtype diffuse astrocytoma group did not show any
molecular genetic aberrations shown above. The study wasapproved by the institutional review boards, and the detailed
information of the cases was provided in Supplementary
Table 1.
2.2. DNA Extraction and Sanger Sequencing. Genomic DNA
was extracted from formalin-fixed paraffin-embedded(FFPE) tissue of the solid tumor regions confirmed on H&E
sections, using QIAamp DNA FFPE Tissue Kit (QIAGEN,
Venlo, The Netherlands), followed by the purification with
NucleoSpin gDNA Clean-up Kit (MACHEREY-NAGEL,
Dusseldorf, Germany), according to the manufacturer’s
instructions. Sequences of the primer sets for polymerase
chain reaction (PCR) were provided in Supplementary
Table 2. PCR was performed using AmpliTaq Gold 360 DNA
Polymerase and GC enhancer (Thermo Fisher Scientific,
Waltham, MA). Sequencing was carried out by Eurofins
Genomics (Tokyo, Japan) with each forward PCR primer asa sequencing primer.
2.3. Molecular Diagnostics. The status of IDH1 (R132H), p53,
and ATRX was analyzed by immunohistochemistry using theautomated immunostaining processor Histostainer (Nichirei
Biosciences, Tokyo, Japan). The antibodies used include antiIDH1 (R132H) (DIA-H09; Dianova, Hamburg, Germany),
anti-p53 (DO-7; Nichirei), and anti-ATRX (HPA001906;
Sigma-Aldrich, St. Louis, MO). We judged the tumor as p53positive when >50% of the tumor nuclei displayed the strong
immunoreactivity of p53, which could be corroborated by theloss of immunoreactivity for ATRX (inactivating mutation)and 1p/19q-nondeletion in the astrocytic tumors [19, 20, 22].
The status of chromosomes 1p and 19q was examined byfluorescence in situ hybridization (FISH) with 1p36 and 19q13
probes (Vysis; Abbott Molecular, Abbott Park, IL), and signal
ratios were calculated according to the previous report [23].
Two reported hot spot mutations (C228T and C250T) in a
TERT promoter region were analyzed by Sanger sequencing.
The hotspot mutations at codon 132 of IDH1 and codon 172
of IDH2 were also screened by Sanger sequencing if the cases

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H&E

IDH1 (R132H)

p53

ATRX

1p

19q

AA

AO

GBM

AOA (IDHwt)(a)

GBM_1 (IDHwt GBM)

Astro_1 (AA)

GBM_3 (IDHwt GBM)

Astro_1 (AA)C C C CC T C C G GC C C CC T C CG GC C CC TC CC G G

CC C C TCCCGG GTERT mutation(C228T)TERT wildtype(C228)TERT mutation(C250T)TERT wildtype(C250)

(b)

100
80

GBM_2 (IDHwt GBM)

Oligo_1 (OL)C GCG C T CCC CAC G C G C T TC CCA CTERT SNP(T349C)TERT SNP(T349)

60
40
20
IDH-wildtype
diffuseastrocytoma

IDH-wildtype
GBM

Oligodendroglial(OL, AO)

0
Astrocytic with
IDH-mutant(DA, AA)

Mutation frequency (%)

TERT promoter mutations

C228T
C250T
(c)

(d)

Figure 1: Molecular profile of the glioma cases. (a) Diffuse astrocytic (DA/AA) tumors were characterized by immunopositivity for IDH1
(R132H) and p53, and the loss of immunoreactivity for ATRX. Oligodendroglial tumors demonstrated IDH1 (R132H) immunoreactivity and1p/19q-codeletion on FISH (red: probes for 1p36 or 19q13, green: probes for the centromere of chromosome 1 or 19). Note that these genotypesof astrocytomas and oligodendrogliomas are mutually exclusive except for IDH genes. IDH-wildtype GBM tumors did not possess mutationsin IDH genes. The IDH-wildtype diffuse astrocytoma group did not show any molecular genetic aberrations shown above. (b) Mutationsat C228 and C250 of the TERT promoter region were examined for every case by Sanger sequencing. Representative cases were shown for
TERT promoter mutations and wildtype TERT. (c) Examination of the frequency of TERT promoter mutations in each group of gliomas.
Oligodendroglial tumors and IDH-wildtype GBM possessed high incidence of TERT mutations, followed by the IDH-wildtype diffuse
astrocytoma. No grade II and III astrocytic tumors with IDH-mutant showed TERT promoter hotspot mutations. (d) Sanger sequencing
results displayed representative cases for TERT promoter common SNPs (T349C). The molecular profile of the cases was summarized inTable 1 and Supplementary Table 1. AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; AOA, anaplastic oligoastrocytoma; DA,
diffuse astrocytoma; GBM, IDH-wildtype glioblastoma; OL, oligodendroglioma; wt, wildtype. Scale bar = 40 𝜇m.

4
did not show immunoreactivity for mutated IDH1 (R132H)[24, 25].
2.4. Cell Lines. U87 malignant glioma cell lines are a kind
gift from Dr. Paul Mischel laboratory (Ludwig Institute forCancer Research, San Diego). Cells were cultured in DMEM
supplemented with 10% FBS (Omega Scientific, Tarzana,CA) in a humidified 5% CO2 incubator at 37∘ C. Analyses
with immunocytochemistry and Sanger sequencing were
performed using the cell lines as described below.
2.5. Development of TERT-Specific Antibody. Human TERT(hTERT) cDNA (Accession number NM 001193376.1) encoding a specific peptide composed of Glu281-Ala436 aminoacid residues was obtained by PCR using a cDNA derived
from the LC-AI or HT1080 cell line as a template. The
primer set for hTERT was as follows: 5󸀠 -AAGGATTTCAGAATTCGAAGCCACCTCTTTGGA-3󸀠 (forward) and5󸀠 -TGCCGTCTCCGAATTCCGCCACAGAGCCCTGGG3󸀠 (reverse). The hTERT-specific peptide was subcloned into
an expression vector, pMAL-c2 (New England Biolabs, Beverly, MA) with MAP tag (GDGMVPPGIEDK) [26], and PA
tag (GVAMPGAEDDVV) [27] using In-Fusion PCR cloning
kit. Competent Escherichia coli (E. coli) TOP-10 cells (Thermo
Fisher Scientific) were transformed with the plasmid, pMALc2MAPhPAter/TERTepi. Then, they were cultured overnightat 37∘ C in LB medium (Thermo Fisher Scientific) containing
100 𝜇g/ml ampicillin (Sigma-Aldrich, St. Louis, MO). Cell
pellets were resuspended in phosphate-buffered saline (PBS;
nacalai tesque, Kyoto, Japan) with 1% Triton X-100 with50 𝜇g/ml aprotinin (Sigma-Aldrich). After sonication, thecrude extracts were collected by centrifugation (9000 ×g,
30 min, 4∘ C). The supernatants were loaded onto Amylose
resin. The loaded resins were washed extensively with column
buffer consisting of 20 mM Tris-HCl (pH 7.4), 200 mM NaCland 1 mM EDTA, and the fusion proteins were eluted bycolumn buffer with 10 mM maltose.
BALB/c mice were immunized by intraperitoneal (i.p.)
injection of the synthetic peptide of hTERT (302–321 aminoacids; Sigma-Aldrich) together with Imject Alum (Thermo
Fisher Scientific). After several additional immunizations,a booster injection was given i.p. two days before splenocytes were harvested. The splenocytes were fused with P3U1
cells obtained from the American Type Culture Collection(ATCC; Manassas, VA) using PEG1500 (Roche Diagnostics,
Indianapolis, IN). The hybridoma cells were grown in Roswell
Park Memorial Institute (RPMI) medium with hypoxanthine,aminopterin, and thymidine selection medium supplement(Thermo Fisher Scientific). The culture supernatants werescreened using direct enzyme-linked immunosorbent assay(ELISA) for the binding to synthetic peptide and recombinant
protein of hTERT purified from E. coli. The Animal Care and
Use Committee of Tohoku University approved the animal
experiments about hybridoma production in this study.
2.6. Verification of the Specificity of Anti-hTERT Antibodies
Using ELISA. Synthetic peptide and recombinant proteinof hTERT were immobilized on Nunc Maxisorp 96-well
microplates (Thermo Fisher Scientific) at a concentration

BioMed Research Internationalof 1 𝜇g/ml and 5 𝜇g/ml, respectively, for 30 min. After
blocking with 1% bovine serum albumin (BSA) containing
0.05% Tween20 in PBS, the plates were incubated with
culture supernatant followed by 1 : 3000 diluted peroxidaseconjugated anti-mouse IgG (Dako, Glostrup, Denmark).
The enzymatic reaction was conducted with a 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific). The optical density
was measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Berkeley, CA).
2.7. Immunoprecipitation-RNA-Dependent RNA Polymerase(IP-RdRP) Assay. 1 × 107 cells treated with nocodazole werelysed in 1 ml of lysis buffer A [0.5% NP-40, 20 mM Tris-HCl(pH 7.4) and 150 mM NaCl]. After sonication, cell lysates werecleared of insoluble material by centrifugation at 21,000 ×g at
4∘ C for 15 min. One milliliter of the lysates was preabsorbed
2 times with 40 𝜇L of Protein L Agarose (Sigma-Aldrich) for
30 min at 4∘ C. The preabsorbed lysates were mixed with 10 𝜇gof an anti-human TERT monoclonal antibody and 40 𝜇L
of Protein L Agarose and incubated overnight at 4∘ C. The
remaining of the IP-RdRP assay was performed as described
previously [28, 29].
2.8. Immunohistochemistry for TERT. All sections were deparaffinized in xylene and rehydrated in an ethanol gradient.
Sections were subjected to antigen retrieval by microwavingin 10 mM citrate buffer (pH 6.0) for 20 min (95∘ C, 500 W).
Endogenous peroxidase activity was quenched with 0.3%
H2 O2 in methanol. The sections were then incubated witha primary antibody [TMab-6; mouse IgM, kappa; diluted
in 1 : 2000 (0.5 𝜇g/mL)] at room temperature for 90 min.
The optimal concentration of the primary antibody wasdetermined by a serial dilution of the antibody. After rinsing,
the sections were subjected to the polymer-immunocomplex
method using EnvisionTM (Dako) for enhancing. 3,3󸀠 Diaminobenzidine tetrahydrochloride (DAB; Dojindo,
Kumamoto, Japan) was used as the chromogen and hematoxylin as the counterstain. To confirm the specificity of the
TERT expression state in glioma cells, negative control reaction was performed by omission of the primary antibodies,and preabsorption test of the antibodies was also conductedusing the synthetic peptide of hTERT (Sigma-Aldrich) at a
final concentration of 0.1 mg/mL. The percentage of TERTpositive cells in each case was semiquantitatively evaluated as
1+ (a few weakly positive cells), 2+ (positive cells < 50%), and3+ (positive cells ≥ 50%). We also immunostained the glioma
samples with the most widely used, commercially available
antibody for TERT (sc-7215; Santa Cruz Biotechnology,
Santa Cruz, CA), but the staining was rather faint than thatby TMab-6 (Supplementary Figure 1). Thus, we evaluatedimmunohistochemical observations of TERT in all theglioma samples with our newly developed TMab-6.
2.9. Western Blotting. Immunoblotting was performed according to a modified version of the previously reported
methods [30, 31]. Snap-frozen tissue samples were lysed andhomogenized with radioimmunoprecipitation assay (RIPA)
lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP-40,
0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate

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Table 1: Molecular information of the cases.

Classification
Astrocytic with IDH-mutant (DA/AA)
Oligodendroglial
GBM
DA/AA with IDH-wildtype
Total

Number
11
10
11
9
41

TERT mt
TERT SNP IDH1/2 mt p53 IHC ATRX IHC 1p/19q-codel
C228T (%) C250T (%) T349C (%)
(%)
(%)
(%)
(%)
0
0
54.5
100
72.7
0
0
70
30
30
100
0
100
100
45.5
18.2
90.9
0
27.3
100
0
33.3
11.1
33.3
0
0
100
0
36.6
14.6
53.7
51.2
26.8
73.2
24.4

AA, anaplastic astrocytoma; codel, codeletion; DA, diffuse astrocytoma; GBM, IDH-wildtype glioblastoma; IHC, immunohistochemistry; mt, mutation.

(SDS)] from Boston BioProducts (Boston, MA). Protein
concentration of each sample was determined using the BCA
kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein extracts wereseparated by electrophoresis on Any kD Mini-PROTEANTGX Precast Gels (Bio-Rad) and then transferred to anitrocellulose membrane with Trans-Blot Turbo Transfer
System (Bio-Rad). The membrane was probed with the primary antibodies against TERT (sc-7215) and glyceraldehyde
phosphate dehydrogenase (GAPDH) (D16H11; Cell Signaling
Technology, Danvers, MA), followed by secondary antibodies conjugated to horseradish peroxidase. TMab-6 was
not available for the immunoblotting study because TMab6 was developed specifically for the immunohistochemical
purpose and produced nonspecific bands on Western blots.
The immunoreactivity was detected with Super Signal West
Pico Chemiluminescent Substrate or West Femto Trial kit(Thermo Fisher Scientific). Quantitative densitometry analysis was performed with image analysis software (ImageJ
version 1.49, NIH).

detecting these two reported hotspot mutations (Figure 1(b)).
As a result, mutations in the TERT promoter were foundin 21 out of 41 tumor samples (51.2%). Consistent withprevious reports [5], oligodendroglial tumors (10 mutations
out of 10 tumors: 100%) and IDH-wildtype GBM (7 mutations
out of 11 tumors: 63.6%) possessed high incidence of TERT
promoter mutations whereas no astrocytic tumors with IDH
mutations showed them (Figure 1(c)). Interestingly, someof the IDH-wildtype diffuse astrocytomas demonstrated
hotspot mutations in the TERT promoter (4 mutations outof 9 tumors: 44.4%). In addition to the TERT promoter
hotspot mutations, we identified a common single nucleotide
polymorphism (SNP) (T349C) [32] across all types of gliomas
we investigated (Figure 1(d)): IDH-mutant astrocytic tumors(6 out of the 11 tumors: 54.5%), oligodendroglial tumors(3 out of the 10 tumors: 30.0%), GBM (10 out of the
11 tumors: 90.9%), and IDH-wildtype diffuse astrocytomas(3 out of the 9 tumors: 33.3%). The molecular profile ofthe cases was summarized in Table 1 and Supplementary
Table 1.

2.10. Quantitative Reverse Transcription Polymerase Chain
Reaction (qRT-PCR) Analysis. Total RNA was extracted by
the use of RNeasy Plus Mini Kit (QIAGEN). Firststrand
cDNA was synthesized by the use of iScript! RT Supermix
for RT-qPCR (Bio-Rad). Real-time RT-PCR was performed
with the SYBR" Premix Ex Taq! II (Tli RNaseH Plus)(Takara, Kyoto, Japan) on Thermal Cycler Dice Real Time
System TP800 (Takara) following the manufacturer’s instructions. 𝛽-Actin was used as an endogenous control. Primer
sequences were provided in Supplementary Table 2.

3.2. Production and Validation of a Novel Anti-TERT Monoclonal Antibody Available for Human Tissue. In order toevaluate the expression of TERT protein in human glioma
tissue, we developed and screened novel anti-hTERT monoclonal antibodies that will be applicable to human tissue
sections. We generated about 10,000 hybridomas from two
mice and picked up about 150 wells by using ELISA. Next, we
screened by using ELISA (synthetic peptide and recombinant
protein), western blotting (recombinant protein and GBM
cell line lysate), and immunohistochemistry. Some clones
were excluded because of nonspecific reactivity by theseanalyses. Finally, we established nine hybridomas as TMabclones. Among these, TMab-6 showed sensitive reactivity in
immunohistochemistry with human tumor tissue of anaplastic oligodendroglioma (IDH-mutant and 1p/19q-codeleted),
glioblastoma (IDH-wildtype), and anaplastic astrocytoma(IDH-mutant). In contrast, the other clones did not show
sensitive and specific reactivity in immunohistochemistry.
We used clone TMab-6 for further studies. Next, we validated
the specificity of TMab-6 by immunoprecipitation-based
assessment of the enzymatic activity of TERT as well as antibody preabsorption test for tissue sections (Figures 2(a) and2(b)). Since TMab-6 was IgM clone, we immunoprecipitatedendogenous TERT complex by protein L beads from HeLa
cells synchronized in mitotic phase by nocodazole [28, 29].
We found that a novel anti-hTERT mAb TMab-6 specifically

2.11. Statistical Analysis. The data were routinely compared
between three or more groups with a two-way analysis ofvariance (ANOVA) followed by post hoc test Bonferroni
corrections for TERT expression using the Statistical Package
for the Social Sciences software package (Version 16.0; SPSS,
Chicago, IL, USA). 𝑝 value below 0.05 was considered
statistically significant.

3. Results

3.1. Molecular Profiling of the Cases. All 41 tumor samples
and 4 control brain samples were subjected to histological aswell as molecular genetic diagnostics, and mutations at C228
and C250 of the TERT promoter region were subsequently
examined for every case by Sanger sequencing, successfully

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BioMed Research International
Protein A

10E9-2

TMab-6

Irrelevant IgM

nt

Preimmune

Protein L

Case: Astro_11 (AA) Case: IDHwt_5 (AOA)(TERT mt C228T)(TERT wt)
Without
TERT primary Ab(TMab-6)

With
TERT primary Ab

150
100
90
80
70
60

With
TERT primary Ab+
Neutralization peptide(Ab : peptide = 1 : 1)

50
40
30

With
TERT primary Ab+
Neutralization peptide(Ab : peptide = 1 : 10)

20

(a)

(b)

U87 malignant glioma cell line
[TERT mt (C228T)]C C C C T T C C GTERT mutation(C228T)(c)

Figure 2: Validation of a newly developed TERT-specific antibody (TMab-6) usable for human glioma tissue. (a) Endogenous TERT was
immunoprecipitated with an anti-human TERT mAb (TMab-6) followed by an RNA-dependent RNA polymerase (RdRP) assay using HeLa
cells treated with nocodazole. Arrows indicate RdRP products. The preimmune (without antibodies) and irrelevant IgM lanes are a negative
control, and the 10E9-2 antibody (MBL, Nagoya, Japan) lane is a positive control. nt, nucleotide. (b) The IDH-mutant astrocytoma (anaplastic
astrocytoma: AA) and IDH-wildtype diffuse astrocytoma (Histologically anaplastic oligoastrocytoma: AOA) cases were analyzed for TERT
protein expression. TMab-6 specifically recognized the nuclei of the tumor cells, and immunoreactivity was not detected in the negative
control section without the application of TMab-6. An antibody absorption test with synthetic neutralization peptide of TERT further
validated the specificity of TMab-6 as an anti-TERT antibody which is applicable to human glioma tissue. (c) TMab-6 Immunostainingof U87 malignant glioma cell lines with a TERT hotspot mutation (C228T) showed strong nuclear immunoreactivity for TERT. Ab, antibody;
mt, mutation. Scale bar = 40 𝜇m.

recognized human TERT protein, and that it could beapplied for immunohistochemistry on the human glioma
tissue sections. We further confirmed the nuclear stainingof TERT by TMab-6 in U87 malignant glioma cell lines(Figure 2(c)) that were previously reported to possess TERT
hotspot mutations as well as upregulated TERT transcripts[5, 33].

3.3. Expression of TERT in Human Glioma Tissue. After confirming the specificity of a newly developed anti-TERT monoclonal antibody TMab-6, we performed immunohistochemical staining on sections of nonneoplastic adult human cerebral tissues and all glioma samples including IDH-mutatedastrocytoma, oligodendroglioma, IDH-wildtype GBM, andIDH-wildtype diffuse astrocytoma and examined the staining

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H&E

TERT

Astro (AA)(TERT wt)

Control (nonneoplastic tissue)
TERT WT

Oligo (AO)
(TERT mt C228T)

Case: Astro_4 (AA)TERT WT

IDHwt GBM(TERT mt C228T)
Case: Oligo_10 (AO)
TERT mt C228T

IDHwt (AA)(TERT wt)

Case: GBM_5 (IDHwt GBM)TERT WT
IDHwt (AOA)(TERT mt C228T)

(a)

(b)

Figure 3: TERT expression in human glioma samples. (a) TMab-6 immunostaining of the glioma samples demonstrated strong nuclear aswell as some cytoplasmic staining in tumor cells. Representative tumors were shown in the astrocytic, oligodendroglial, IDH-wildtype GBM,
and IDH-wildtype diffuse astrocytoma groups. Glioma cells in all the cases showed nuclear immunostaining of TERT to a various degree.
(b) TMab-6 immunostaining of vascular endothelial cells (arrows) in the nonneoplastic cerebral tissues, TERT-wildtype AA, TERT-mutated
AO, and TERT-mutated GBM. AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; AOA, anaplastic oligoastrocytoma; GBM,
IDH-wildtype glioblastoma; mt, mutation; wt/WT, wildtype. Scale bar = 40 𝜇m.

patterns of TERT protein in them. Immunohistochemical
observations using TMab-6 demonstrated strong staining in
the nucleus and some cytoplasmic staining of the glioma
cells (Figure 3(a)), and TERT immunoreactivity was notobserved in the normal tissue adjacent to the tumor tissue.
Surprisingly, glioma cells in all the cases showed nuclear
immunostaining for TERT to various degrees compared withthe cells in nonneoplastic tissues (Figure 3(a)) althoughsome intratumoral heterogeneity of the nuclear staining wasobserved. Some reactive astrocytes in cerebral parenchyma
from the cases with hippocampal sclerosis and corticaldysplasia showed nonspecific, weak cytoplasmic staining(Supplementary Figure 2). Interestingly, endothelial cells of
the tumor vessels also displayed nuclear immunostaining
in both TERT-mutated and wildtype tumors, whereas vascular endothelial cells in the nonneoplastic tissues did notshow any specific TERT immunoreactivity in their nuclei(Figure 3(b)).

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3.4. Semiquantitative Evaluation of TERT Expression Associated with TERT Promoter Mutations in Human Gliomas.
Having confirmed the findings that all the glioma cells
we analyzed showed nuclear immunostaining of TERT tovarious degrees, we further examined the association of
the TERT promoter mutations with TERT immunoreactivity
in our glioma samples. Unexpectedly, immunohistochemistry using TMab-6 demonstrated that immunoreactivity for
TERT did not correspond to the mutational status of the
TERT promoter region across glioma samples (Figure 4(a)).
Although the staining was weak, nuclear immunoreactivity
for TERT was also confirmed in both TERT-wildtype andmutated gliomas with the commercially available antibody
for TERT (sc-7215) (Supplementary Figure 1). In addition
to the hotspot mutations in the TERT promoter (C228T
and C250T), the presence of a common SNP in the TERT
promoter (T349C) was not associated with TERT immunoreactivity (Supplementary Table 1). TERT immunohistochemistry was not capable of identifying the differences between
TERT-mutated gliomas and TERT-nonmutant gliomas (Figure 4(a)). The scores might be relevant to tumor grading
and aggressiveness represented by IDH-wildtype GBMs andIDH-wildtype diffuse astrocytomas (Figure 4(a)), both ofwhich behave aggressively with a dismal prognosis [1, 4, 14,21]. Finally, the relationship between the TERT promoter
mutations, the expression levels of TERT protein, and tumor
entity was further verified by Western blotting (Figure 4(b)),
and the level of TERT mRNA was not parallel with its mutational status across all the glioma samples (data not shown).

4. Discussion
The mechanism of TERT upregulation in cancer has largely
remained unknown to date. Recurrent mutations at two
hotspot regions (C228T and C250T) in the TERT promoter
have been reported in various types of cancers [2, 3, 5],
and a recent report outlined the mechanism that TERT
promoter mutations reactivate TERT expression [34]. In
diffuse gliomas, the sole mechanism to upregulate TERTmRNA is considered to be two hotspot mutations in the
TERT promoter region. However, the level of TERT transcripts varies widely in expression among tumors including
gliomas [5, 7], skin cancers [2, 3, 35], thyroid cancers [7],hepatocellular carcinomas [36], bladder cancers [37], andmalignant lymphomas [38]. The levels of TERT mRNA tend
to be increased in TERT-mutated tumors in comparison
with TERT-wildtype tumors, but it is not necessarily thecase for the individual tumors, indicating that mutationsin the TERT promoter alone could not explain the wide
variety in the expression of TERT transcripts. Further, tothe best of our knowledge, only one report has so far
investigated the expression of TERT protein in gliomas, andany difference was not detected between TERT-mutated and
TERT-wildtype GBMs [7].
In the present study, we compared the expression of TERT
protein with its mutational status in adult gliomas. In linewith the previous studies, TERT expression was confirmedin the nuclei of oligodendroglial tumors and IDH-wildtype
GBM which possess either of two hotspot mutations in the

BioMed Research International
promoter regions of TERT. Surprisingly, however, even IDHmutant astrocytic tumors (DA/AA) without known hotspot
mutations in the TERT promoter displayed certain amountof TERT immunoreactivity in their nuclei. Additionally, qRTPCR study did not demonstrate any significant correlation
between TERT promoter mutations and the level of TERTmRNA expression. Considering our findings that immunoprecipitation with TMab-6 did demonstrate the same blotting
bands as the commercially available antibody (10E9-2/sc7215) and TMab-6 and sc-7215 showed the similar immunostaining pattern in the tumor, TERT immunoreactivity issurely increased in tumor tissue in comparison with normal tissue although the possibility of any unknown crossreactivity cannot be completely ruled out. The finding that
the TERT immunoreactivity in endothelial as well as tumor
cells is strictly limited to the neoplastic tissue further supports
the idea that TMab-6 detects TERT protein closely bounded
to the tumor environment composed of neoplastic tissue andneovasculatures.
Our findings of an unexpected increase in the expressionof TERT protein even in the TERT-wildtype gliomas raise thepossibility that the expression of TERT mRNA and protein
could be regulated by the mechanism other than its promoter
mutations, including via epigenetic factors. In addition toreported hotspot mutations, certain SNPs in the TERT
promoter region (T349C) were reported to suppress TERTtranscripts [32], but we could not confirm any associationof SNPs with TERT protein expression, further supporting
our hypothesis that TERT expression could be epigeneticallyregulated.
There could be several mechanisms to increase theexpression of TERT protein in TERT-wildtype tumors. It has
been reported that DNA hypermethylation of the TERT promoter is associated with TERT upregulation in pediatric brain
tumors [8] and other tumors [39], and histone acetylation
at the TERT promoter regions may also be relevant to its
expression [9]. IDH-mutant diffuse astrocytic tumors often
demonstrate the glioma-CpG island methylator phenotype(G-CIMP) [40, 41], and the nuclear TERT staining in diffuse
astrocytomas might be epigenetically caused by hypermethylation of the TERT promoter [10]. Additionally, a numberof human tumors maintain their telomeres by a telomeraseindependent mechanism termed alternative lengthening oftelomeres (ALT) caused by such abnormalities as loss of
ATRX [42, 43], and this might somehow affect the expression
level of TERT.
In addition to its applicability to the molecular diagnostics of gliomas, TERT could be a good therapeutic target
against diffuse gliomas which are a potentially malignant,incurable brain tumor in human. A novel TERT-targetingtherapy [44, 45] would be expected to specifically target
IDH-wildtype GBM including primary brain tumor initiating cells and 1p/19q-codeleted oligodendroglioma whichhave characteristically possessed hotspot mutations in the
TERT promoter region [5, 33]. Our unexpected findings
suggest that a specific TERT-targeting therapy might be a
promising therapeutic strategy against all types of gliomas bydirectly targeting tumor cells with an indirect antiangiogenic
effect on tumor vasculature like the antiangiogenic agent

BioMed Research International9#3 (OL)#5 (GBM)#4 (AO)#6 (GBM)

C250T

3+

C250T

TERT immunoreactivity
p = 0.09

p = 0.07

p = .

p = .2

IDH-wildtype
diffuseastrocytoma

TERT IHC score

2+

C250T

4

1+

#8 (OA)IDH wildtype
GBM

WT

WT

Oligodendroglial(OL, AO)#2 (AA)

Score

C250T

C250T

WT

#7 (AOA)

Astrocytic with
IDH-mutant(DA, AA)#1 (DA)

3
2
1

Nonneoplastic(HS, CD)

0

(a)

10

TERT
(LE)
GAPDH

4
2
0
A#1

38
(kDa)

∗

6

IDHwt#8

52

13.4

8

GB#5

102
76

TERT
(120 kDa)

OL#4

∗

23.9

IDHwt#7

IDHwt DA

OL#3

GBM

#1 #2 #3 #4 #5 #6 #7 #8
Relative expression

225
150

Oligo

A#2

Astro

GB#6

TERT protein expression(b)

Figure 4: Association of TERT promoter mutations and its immunoreactivity in human gliomas. (a) Immunostaining of TERT by TMab-6
demonstrated that TERT immunoreactivity did not correspond to the mutational status of the TERT promoter region across glioma samples.
Semiquantitative analysis of the TERT-immunostaining, scoring the staining intensities as 1+ (a few weakly positive cells), 2+ (positive cells <50%), and 3+ (positive cells ≥ 50%). Blue dots represent TERT-wildtype cases and red denotes TERT-mutated cases. 𝑃 values for nonneoplastic
tissue versus each glioma entity. (b) An analysis on the relationship of the TERT promoter mutations, the expression level of TERT protein
and each tumor entity by Western blotting with the most widely used, commercially available TERT antibody (sc-7215). A bar graph showed
the quantification of TERT expression in each case, normalized by a loading control GAPDH expression. ∗ Nonspecific bands. LE, longexposure. AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; AOA, anaplastic oligoastrocytoma; CD, cortical dysplasia; DA,
diffuse astrocytoma; GBM, IDH-wildtype glioblastoma; HS, hippocampal sclerosis; OA, oligoastrocytoma; OL, oligodendroglioma; IDHwt,
IDH-wildtype diffuse astrocytoma; wt/WT, wildtype. Scale bar = 40 𝜇m.

bevacizumab, a humanized monoclonal antibody against
vascular endothelial growth factor (VEGF) ligands [46], but
the additional studies are essential to demonstrate that thepresence of TERT protein actually implies more aggressive

biology for the recommendation of treatment based on TERT
immunohistochemistry.
The limitation of our study is that we have not directly
measured the telomerase activity itself. Indeed, some reports

10

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demonstrated that TERT expression and its biological activity
are not necessarily parallel [12]. Further, there could be thepossibility that this novel antibody is picking up baseline
TERT protein that may not be associated with increased
telomerase efficiency or activity. It would be thus importantto further validate/specify the antibody and analyze therelationship between histological TERT immunoreactivity
and its enzymatic activity as a next step. Future studies
are also necessary to unravel the contributing factors in
upregulating the protein expression of TERT to examine if TERT protein expression could be the biomarker
for the applicability of TERT-targeting therapeutics in the
future.
Together, we developed a novel TERT-specific antibody,and it is sensitively and specifically applicable to immunohistochemistry for TERT in human glioma tissue. Immunohistochemistry with this TERT-specific antibody could not playthe diagnostic surrogate for TERT promoter mutations, but
we could find an unexpected increase in TERT immunoreactivity across all types of gliomas and tumor vasculature.
This study is the first extensive analysis on the expressionof TERT protein in human glioma tissue and suggests that
TERT expression could be regulated by various mechanisms
including epigenetic modifications. Future studies with themeasurement of TERT activity could be useful for facilitating
our understanding of telomere biology in cancer including
gliomas.

Supplementary Materials

Disclosure[2] S. Horn, A. Figl, P. S. Rachakonda et al., “TERT promoter
mutations in familial and sporadic melanoma,” Science, vol. 339,
no. 6122, pp. 959–961, 2013.

A part of this work was presented at 11th European Congressof Neuropathology (July 6–9, 2016, Bordeaux, France) and13th Asian Society for Neuro-Oncology (September 11–14,
2016, Sydney, Australia).

Conflicts of Interest
The authors declare no conflicts of interest.

Authors’ Contributions
K. Masui and T. Komori contributed equally to the work.

Acknowledgments
The authors would like to thank Mr. Mizuho Karita,
Mr. Takashi Sakayori (Tokyo Women’s Medical University), and Dr. Mami Yasukawa (Division of Cancer StemCell, National Cancer Center Research Institute) for their
excellent technical support. This research was supported inpart by AMED under Grant nos. JP17am0301010 (YukinariKato), JP17am0101078 (Yukinari Kato), and JP17ae0101028(Yukinari Kato), by JSPS KAKENHI Grant no. 17K07299(Mika K. Kaneko) and Grant no. 16K10748 (Yukinari Kato),and by the Cell Science Research Foundation, Grant-inAid from the Tokyo Biochemical Research Foundation,
and JSPS KAKENHI Grant nos. 15K19067 and 17K15672(Kenta Masui).

Supplementary Table 1. Detailed information of the cases.
Supplementary Table 2. Primer information.
Supplementary Figure 1. Immunostaining of TERT protein
in human gliomas with TMab-6 and sc-7215. We immunostained TERT-wildtype and mutant gliomas with our newly
developed TMab-6 and the most widely used, commercially available sc-7215. Both antibodies detected nuclear
immunoreactivity of TERT in TERT-wildtype and mutatedgliomas although the staining intensities were variable. AA,
anaplastic astrocytoma; AO, anaplastic oligodendroglioma.
Scale bar = 40 𝜇m.
Supplementary Figure 2. Immunostaining of TERT protein
in human nonneoplastic brain tissue. We immunostained thecases of TERT-wildtype hippocampal sclerosis and corticaldysplasia with our newly developed TMab-6 (0.5 𝜇g/mL).
Some reactive astrocytes showed nonspecific, weak cytoplasmic staining although any nuclear staining of TERT was notobserved in them.

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