Elevated TERT Expression in TERT-Wildtype Adult Diffuse ...downloads.hindawi.com/journals/bmri/2018/7945845.pdf · ResearchArticle Elevated TERT Expression in TERT-Wildtype Adult

Research ArticleElevated TERT Expression in TERT-Wildtype AdultDiffuse Gliomas Histological Evaluation with a NovelTERT-Specific Antibody

Kenta Masui1 Takashi Komori 2 Yukinari Kato3 Kenkichi Masutomi4

Koichi Ichimura5 Satoshi Ogasawara3 Mika K Kaneko3 Hiroharu Oki3

Hiroyoshi Suzuki6 Masayuki Nitta7 Takashi Maruyama7 Yoshihiro Muragaki7

Takakazu Kawamata7 Tatsuo Sawada1 and Noriyuki Shibata1

1Department of Pathology Tokyo Womenrsquos Medical University Tokyo 162-8666 Japan2Department of Neuropathology Tokyo Metropolitan Neurological Hospital Tokyo 183-0042 Japan3Department of Antibody Drug Development Tohoku University Graduate School of Medicine Miyagi 980-8575 Japan4Division of Cancer Stem Cell National Cancer Center Research Institute Tokyo 104-0045 Japan5Division of Brain Tumor Translational Research National Cancer Center Research Institute Tokyo 104-0045 Japan6Department of Pathology and Laboratory Medicine Sendai Medical Center Miyagi 983-0045 Japan7Department of Neurosurgery Tokyo Womenrsquos Medical University Tokyo 162-8666 Japan

Correspondence should be addressed to Takashi Komori komori-tkigakukenorjp

Received 16 August 2017 Revised 10 December 2017 Accepted 17 January 2018 Published 5 March 2018

Academic Editor Jens Schittenhelm

Copyright copy 2018 Kenta Masui et al This is an open access article distributed under the Creative Commons Attribution Licensewhich 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 selectivelyobserved among 1p19q-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 rarelyinvestigated thus far It would be thus critical to examine the expression level of TERT in tumors in addition to its mutationalstatus and sensitive and specific methods are urgently needed to examine TERT protein expression for the assessment of TERTbiology in gliomas Using our newly developedTERT-specificmonoclonal 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 proteinexpression 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 prognosesbased on the status of isocitrate dehydrogenase genes 1 and2 (IDH12) mutations and 1p19q-codeletion [1] Recentlyhotspot mutations in the telomerase reverse transcriptasegene (TERT) promoter (chromosome 5 1295228 CgtT and1295250 CgtT) have been identified in melanomas [2 3]Thesubsequent investigation in a series of gliomas demonstratedthe applicability of the TERT promoter mutations in theclassification of diffuse gliomas [4] and the selectively high

frequency of the TERT promoter mutations was observedamong 1p19q-codeleted oligodendrogliomas and IDH-wildtype glioblastoma (GBM) [5] The presence of TERTpromoter mutations is well-correlated with three distinctiveglioma groups [1] suggesting that TERT could be deeplyinvolved in the biology of diffuse gliomas

Hotspot mutations in the TERT promoter (C228T andC250T) are currently recognized as the only event in gliomasto upregulate TERT transcripts and mRNA of TERT tendsto 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

HindawiBioMed Research InternationalVolume 2018 Article ID 7945845 12 pageshttpsdoiorg10115520187945845

2 BioMed Research International

in various cancers including gliomas [5 7] and there maybe other potential mechanisms to upregulate TERT mRNAin addition to its promoter mutations Indeed in other braintumors and systemic cancers epigenetic shifts in the TERTpromoter regions including DNA methylation and histonemodifications were reported to regulate TERT transcriptsand reactivation [8ndash10] necessitating a rethinking of themechanism to upregulate the expression of TERT

As an essential component of telomerase TERT addstelomeric repeats to chromosomal ends using telomeraseRNA component (TERC) as a template and maintainstelomere length Telomerase activity had been considered tobe maintained in most types of the cancer cells enablingthem to proliferate and survive indefinitely [11] However arecent report demonstrated that telomere length is positivelycorrelated with mutations in the alpha thalassemiamentalretardation syndrome X-linked gene (ATRX) but not theTERT promoter suggesting that TERT upregulation itselfmay be essential for glioma biology irrespective of telomerelength [12] It would be thus critical to examine the expressionlevel of TERT protein in diffuse gliomas to clarify the role ofTERT in their biology in addition to its mRNA level as wellas mutational status but few reports have so far validatedthe expression of TERT protein in human glioma tissue[7] Sensitive and specific methods are strongly desired toexamine TERT protein expression for the future analyses onthe TERT biology in diffuse gliomas

In this study we developed a TERT-specific antibodyapplicable to human tissue and examined the expression ofTERT in a series of glioma samples Although the practicaldiagnostic utility of the antibody to detect the TERT-mutatedtumors was expected TERT immunohistochemistry wasnot capable of identifying the differences between TERT-mutated gliomas and TERT-nonmutant gliomas Surpris-ingly however an increase in TERT protein expressionwas detected across all types of gliomas including tumorswith wildtype TERT and the level of TERT expressionwas different even among the TERT-mutant gliomas suchas oligodendrogliomas and GBMs Our study is the firstthorough analysis on the expression of TERT protein using aspecific TERT monoclonal antibody in human glioma tissuesuggesting that TERT protein expressionmay be regulated byseveral mechanisms in addition to its promoter mutation

2 Materials and Methods

21 Tissue Collection A total of 41 human glioma tissuesand 4 nonneoplastic adult human cerebral tissues fromfour epileptic patients were obtained at surgery from TokyoWomenrsquos Medical University Hospital and Tokyo Metropoli-tan Neurological Hospital The tumor samples included 11grade II and III astrocytic tumors with IDH mutations[diffuse astrocytoma (DA) and anaplastic astrocytoma (AA)]10 grade II and III oligodendroglial tumors 11 grade IVIDH-wildtype GBMs and 9 IDH-wildtype diffuse astrocy-tomas including tumors histologically diagnosed as diffuseastrocytoma anaplastic astrocytoma oligoastrocytoma andanaplastic oligoastrocytoma which did not show IDH muta-tions p53 immunopositivity and the loss of ATRX expression

(wtIDHp53-ATRX+) Clinical information of the cases wascollected from the medical records All specimens werefixed in 10 buffered formalin and embedded into paraffinsections Sections were routinely stained with hematoxylinand eosin (HampE) and histopathological diagnosesweremadeaccording to the World Health Organization (WHO) 2016classification of tumors of the nervous system [13ndash15] andInternational Society of Neuropathology-Haarlem consensusguidelines [16]

All tumor and control brain samples were subjected tohistological diagnosis followed by molecular genetics screen-ing for a more precisemolecular diagnosis (Figure 1(a)) [17]Tumors in the astrocytoma group (DAAA) were charac-terized by detectable immunoreactivity for mutated IDH1(R132H) and p53 [18] and the loss of immunoreactivity forATRX [19 20] All tumors in the oligodendroglioma groupdemonstrated mutated IDH1 (R132H) and 1p19q-codeletionGBM tumors did not possess mutations in IDH correspond-ing to ldquoGBM IDH-wildtyperdquo [14 21] The tumors in theIDH-wildtype diffuse astrocytoma group did not show anymolecular genetic aberrations shown above The study wasapproved by the institutional review boards and the detailedinformation of the cases was provided in SupplementaryTable 1

22 DNA Extraction and Sanger Sequencing Genomic DNAwas extracted from formalin-fixed paraffin-embedded(FFPE) tissue of the solid tumor regions confirmed on HampEsections using QIAamp DNA FFPE Tissue Kit (QIAGENVenlo The Netherlands) followed by the purification withNucleoSpin gDNA Clean-up Kit (MACHEREY-NAGELDusseldorf Germany) according to the manufacturerrsquosinstructions Sequences of the primer sets for polymerasechain reaction (PCR) were provided in SupplementaryTable 2 PCR was performed using AmpliTaq Gold 360 DNAPolymerase and GC enhancer (Thermo Fisher ScientificWaltham MA) Sequencing was carried out by EurofinsGenomics (Tokyo Japan) with each forward PCR primer asa sequencing primer

23 Molecular Diagnostics The status of IDH1 (R132H) p53and ATRXwas analyzed by immunohistochemistry using theautomated immunostaining processor Histostainer (NichireiBiosciences Tokyo Japan)The antibodies used include anti-IDH1 (R132H) (DIA-H09 Dianova Hamburg Germany)anti-p53 (DO-7 Nichirei) and anti-ATRX (HPA001906Sigma-Aldrich St Louis MO) We judged the tumor as p53-positive when gt50 of the tumor nuclei displayed the strongimmunoreactivity of p53 which could be corroborated by theloss of immunoreactivity for ATRX (inactivating mutation)and 1p19q-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 19q13probes (Vysis Abbott Molecular Abbott Park IL) and signalratios were calculated according to the previous report [23]Two reported hot spot mutations (C228T and C250T) in aTERT promoter region were analyzed by Sanger sequencingThe hotspot mutations at codon 132 of IDH1 and codon 172of IDH2 were also screened by Sanger sequencing if the cases

BioMed Research International 3

AA

AO

GBM

IDH1 (R132H) p53 ATRX 1pHampE 19q

AOA (IDHwt)

(a)

TERT mutation(C228T)

Astro_1 (AA)

TERT wildtype(C228)

GBM_1 (IDHwt GBM)


GBM_3 (IDHwt GBM) Astro_1 (AA)

TERT wildtype(C250)

T T T TC C C C C C C G G GGC CCCCCC C C C C C C C G G GGGCCCC C C C

(b)

0

20

40

60

80

100

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

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(OL

AO

)

IDH

-wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a

TERT promoter mutations

C228TC250T

Mut

atio

n fre

quen

cy (

)

(c)

TERT SNP(T349C)

GBM_2 (IDHwt GBM) Oligo_1 (OL)

TERT SNP(T349)

C CC C CG C G C T A C G C C C CG C T T A C

(d)Figure 1 Molecular profile of the glioma cases (a) Diffuse astrocytic (DAAA) tumors were characterized by immunopositivity for IDH1(R132H) and p53 and the loss of immunoreactivity for ATRX Oligodendroglial tumors demonstrated IDH1 (R132H) immunoreactivity and1p19q-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 forTERT promoter mutations and wildtype TERT (c) Examination of the frequency of TERT promoter mutations in each group of gliomasOligodendroglial tumors and IDH-wildtype GBM possessed high incidence of TERT mutations followed by the IDH-wildtype diffuseastrocytoma No grade II and III astrocytic tumors with IDH-mutant showed TERT promoter hotspot mutations (d) Sanger sequencingresults 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 DAdiffuse astrocytoma GBM IDH-wildtype glioblastoma OL oligodendroglioma wt wildtype Scale bar = 40 120583m


did not show immunoreactivity for mutated IDH1 (R132H)[24 25]

24 Cell Lines U87 malignant glioma cell lines are a kindgift from Dr Paul Mischel laboratory (Ludwig Institute forCancer Research San Diego) Cells were cultured in DMEMsupplemented with 10 FBS (Omega Scientific TarzanaCA) in a humidified 5 CO2 incubator at 37∘C Analyseswith immunocytochemistry and Sanger sequencing wereperformed using the cell lines as described below

25 Development of TERT-Specific Antibody Human TERT(hTERT) cDNA (Accession numberNM 0011933761) encod-ing a specific peptide composed of Glu281-Ala436 aminoacid residues was obtained by PCR using a cDNA derivedfrom the LC-AI or HT1080 cell line as a template Theprimer set for hTERT was as follows 51015840-AAGGATTTC-AGAATTCGAAGCCACCTCTTTGGA-31015840 (forward) and51015840-TGCCGTCTCCGAATTCCGCCACAGAGCCCTGGG-31015840 (reverse) The hTERT-specific peptide was subcloned intoan expression vector pMAL-c2 (New England Biolabs Bev-erly MA) with MAP tag (GDGMVPPGIEDK) [26] and PAtag (GVAMPGAEDDVV) [27] using In-Fusion PCR cloningkit Competent Escherichia coli (E coli) TOP-10 cells (ThermoFisher Scientific) were transformed with the plasmid pMAL-c2MAPhPAterTERTepi Then they were cultured overnightat 37∘C in LB medium (Thermo Fisher Scientific) containing100 120583gml ampicillin (Sigma-Aldrich St Louis MO) Cellpellets were resuspended in phosphate-buffered saline (PBSnacalai tesque Kyoto Japan) with 1 Triton X-100 with50 120583gml aprotinin (Sigma-Aldrich) After sonication thecrude extracts were collected by centrifugation (9000timesg30min 4∘C) The supernatants were loaded onto AmyloseresinThe loaded resins werewashed extensively with columnbuffer consisting of 20mM Tris-HCl (pH 74) 200mMNaCland 1mM EDTA and the fusion proteins were eluted bycolumn buffer with 10mMmaltose

BALBc mice were immunized by intraperitoneal (ip)injection of the synthetic peptide of hTERT (302ndash321 aminoacids Sigma-Aldrich) together with Imject Alum (ThermoFisher Scientific) After several additional immunizationsa booster injection was given ip two days before spleno-cytes were harvested The splenocytes were fused with P3U1cells obtained from the American Type Culture Collection(ATCC Manassas VA) using PEG1500 (Roche DiagnosticsIndianapolis IN)Thehybridoma cells were grown inRoswellParkMemorial Institute (RPMI)mediumwith hypoxanthineaminopterin 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 recombinantprotein of hTERT purified from E coli The Animal Care andUse Committee of Tohoku University approved the animalexperiments about hybridoma production in this study

26 Verification of the Specificity of Anti-hTERT AntibodiesUsing ELISA Synthetic peptide and recombinant proteinof hTERT were immobilized on Nunc Maxisorp 96-wellmicroplates (Thermo Fisher Scientific) at a concentration

of 1 120583gml and 5120583gml respectively for 30min Afterblocking with 1 bovine serum albumin (BSA) containing005 Tween20 in PBS the plates were incubated withculture supernatant followed by 1 3000 diluted peroxidase-conjugated anti-mouse IgG (Dako Glostrup Denmark)The enzymatic reaction was conducted with a 1-Step UltraTMB-ELISA (Thermo Fisher Scientific) The optical densitywas measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories Berkeley CA)

27 Immunoprecipitation-RNA-Dependent RNA Polymerase(IP-RdRP) Assay 1 times 107 cells treated with nocodazole werelysed in 1ml of lysis buffer A [05 NP-40 20mM Tris-HCl(pH 74) and 150mMNaCl] After sonication cell lysates werecleared of insoluble material by centrifugation at 21000timesg at4∘C for 15min One milliliter of the lysates was preabsorbed2 times with 40120583L of Protein L Agarose (Sigma-Aldrich) for30min at 4∘CThe preabsorbed lysates weremixed with 10 120583gof an anti-human TERT monoclonal antibody and 40 120583Lof Protein L Agarose and incubated overnight at 4∘C Theremaining of the IP-RdRP assay was performed as describedpreviously [28 29]

28 Immunohistochemistry for TERT All sections were de-paraffinized in xylene and rehydrated in an ethanol gradientSections were subjected to antigen retrieval by microwavingin 10mM citrate buffer (pH 60) for 20min (95∘C 500W)Endogenous peroxidase activity was quenched with 03H2O2in methanol The sections were then incubated with

a primary antibody [TMab-6 mouse IgM kappa dilutedin 1 2000 (05120583gmL)] at room temperature for 90minThe optimal concentration of the primary antibody wasdetermined by a serial dilution of the antibody After rinsingthe sections were subjected to the polymer-immunocomplexmethod using EnvisionTM (Dako) for enhancing 331015840-Diaminobenzidine tetrahydrochloride (DAB DojindoKumamoto Japan) was used as the chromogen and hema-toxylin as the counterstain To confirm the specificity of theTERT expression state in glioma cells negative control reac-tion was performed by omission of the primary antibodiesand preabsorption test of the antibodies was also conductedusing the synthetic peptide of hTERT (Sigma-Aldrich) at afinal concentration of 01mgmL The percentage of TERT-positive cells in each case was semiquantitatively evaluated as1+ (a few weakly positive cells) 2+ (positive cells lt 50) and3+ (positive cells ge 50)We also immunostained the gliomasamples with the most widely used commercially availableantibody for TERT (sc-7215 Santa Cruz BiotechnologySanta 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

29 Western Blotting Immunoblotting was performed ac-cording to a modified version of the previously reportedmethods [30 31] Snap-frozen tissue samples were lysed andhomogenized with radioimmunoprecipitation assay (RIPA)lysis buffer [50mM Tris-HCl 150mM NaCl 1 NP-4005 sodium deoxycholate and 01 sodium dodecyl sulfate


Table 1 Molecular information of the cases

Classification Number TERT mt TERT SNP IDH12 mt p53 IHC ATRX IHC 1p19q-codelC228T () C250T () T349C () () () () ()

Astrocytic with IDH-mutant (DAAA) 11 0 0 545 100 727 0 0Oligodendroglial 10 70 30 30 100 0 100 100GBM 11 455 182 909 0 273 100 0DAAA with IDH-wildtype 9 333 111 333 0 0 100 0Total 41 366 146 537 512 268 732 244AA anaplastic astrocytoma codel codeletion DA diffuse astrocytoma GBM IDH-wildtype glioblastoma IHC immunohistochemistry mt mutation

(SDS)] from Boston BioProducts (Boston MA) Proteinconcentration of each sample was determined using the BCAkit (Thermo Fisher Scientific) according to the manufac-turerrsquos 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 TransferSystem (Bio-Rad) The membrane was probed with the pri-mary antibodies against TERT (sc-7215) and glyceraldehydephosphate dehydrogenase (GAPDH) (D16H11 Cell SignalingTechnology Danvers MA) followed by secondary anti-bodies conjugated to horseradish peroxidase TMab-6 wasnot available for the immunoblotting study because TMab-6 was developed specifically for the immunohistochemicalpurpose and produced nonspecific bands on Western blotsThe immunoreactivity was detected with Super Signal WestPico Chemiluminescent Substrate or West Femto Trial kit(Thermo Fisher Scientific) Quantitative densitometry anal-ysis was performed with image analysis software (ImageJversion 149 NIH)

210 Quantitative Reverse Transcription Polymerase ChainReaction (qRT-PCR) Analysis Total RNA was extracted bythe use of RNeasy Plus Mini Kit (QIAGEN) FirststrandcDNA was synthesized by the use of iScript RT Supermixfor RT-qPCR (Bio-Rad) Real-time RT-PCR was performedwith the SYBR Premix Ex Taq II (Tli RNaseH Plus)(Takara Kyoto Japan) on Thermal Cycler Dice Real TimeSystem TP800 (Takara) following themanufacturerrsquos instruc-tions 120573-Actin was used as an endogenous control Primersequences were provided in Supplementary Table 2

211 Statistical Analysis The data were routinely comparedbetween three or more groups with a two-way analysis ofvariance (ANOVA) followed by post hoc test Bonferronicorrections for TERT expression using the Statistical Packagefor the Social Sciences software package (Version 160 SPSSChicago IL USA) 119901 value below 005 was consideredstatistically significant

3 Results

31 Molecular Profiling of the Cases All 41 tumor samplesand 4 control brain samples were subjected to histological aswell as molecular genetic diagnostics and mutations at C228and C250 of the TERT promoter region were subsequentlyexamined for every case by Sanger sequencing successfully

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 (512) Consistent withprevious reports [5] oligodendroglial tumors (10 mutationsout of 10 tumors 100) and IDH-wildtypeGBM(7mutationsout of 11 tumors 636) possessed high incidence of TERTpromoter mutations whereas no astrocytic tumors with IDHmutations showed them (Figure 1(c)) Interestingly someof the IDH-wildtype diffuse astrocytomas demonstratedhotspot mutations in the TERT promoter (4 mutations outof 9 tumors 444) In addition to the TERT promoterhotspot mutations we identified a common single nucleotidepolymorphism (SNP) (T349C) [32] across all types of gliomaswe investigated (Figure 1(d)) IDH-mutant astrocytic tumors(6 out of the 11 tumors 545) oligodendroglial tumors(3 out of the 10 tumors 300) GBM (10 out of the11 tumors 909) and IDH-wildtype diffuse astrocytomas(3 out of the 9 tumors 333) The molecular profile ofthe cases was summarized in Table 1 and SupplementaryTable 1

32 Production and Validation of a Novel Anti-TERT Mon-oclonal Antibody Available for Human Tissue In order toevaluate the expression of TERT protein in human gliomatissue we developed and screened novel anti-hTERT mon-oclonal antibodies that will be applicable to human tissuesections We generated about 10000 hybridomas from twomice and picked up about 150 wells by using ELISA Next wescreened by using ELISA (synthetic peptide and recombinantprotein) western blotting (recombinant protein and GBMcell line lysate) and immunohistochemistry Some cloneswere excluded because of nonspecific reactivity by theseanalyses Finally we established nine hybridomas as TMabclones Among these TMab-6 showed sensitive reactivity inimmunohistochemistry with human tumor tissue of anaplas-tic oligodendroglioma (IDH-mutant and 1p19q-codeleted)glioblastoma (IDH-wildtype) and anaplastic astrocytoma(IDH-mutant) In contrast the other clones did not showsensitive and specific reactivity in immunohistochemistryWe used clone TMab-6 for further studies Next we validatedthe specificity of TMab-6 by immunoprecipitation-basedassessment of the enzymatic activity of TERT as well as anti-body 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 HeLacells synchronized in mitotic phase by nocodazole [28 29]We found that a novel anti-hTERT mAb TMab-6 specifically


Protein L Protein A

Prei

mm

une

Irre

leva

nt Ig

M

TMab

-6

10

E9-2

nt

150

100908070

60

50

40

30

20

(a)

WithoutTERT primary Ab

(TMab-6)

WithTERT primary Ab

Case IDHwt_5 (AOA)(TERT mt C228T)

Case Astro_11 (AA)(TERT wt)

WithTERT primary Ab

+Neutralization peptide

(Ab peptide = 1 1)

WithTERT primary Ab

+Neutralization peptide(Ab peptide = 1 10)

(b)

U87 malignant glioma cell line[TERT mt (C228T)]

G

(C228T)TERT mutation

C C CCCC TT

(c)

Figure 2 Validation of a newly developed TERT-specific antibody (TMab-6) usable for human glioma tissue (a) Endogenous TERT wasimmunoprecipitated with an anti-human TERT mAb (TMab-6) followed by an RNA-dependent RNA polymerase (RdRP) assay using HeLacells treated with nocodazole Arrows indicate RdRP products The preimmune (without antibodies) and irrelevant IgM lanes are a negativecontrol and the 10E9-2 antibody (MBL Nagoya Japan) lane is a positive control nt nucleotide (b)The IDH-mutant astrocytoma (anaplasticastrocytoma AA) and IDH-wildtype diffuse astrocytoma (Histologically anaplastic oligoastrocytoma AOA) cases were analyzed for TERTprotein expression TMab-6 specifically recognized the nuclei of the tumor cells and immunoreactivity was not detected in the negativecontrol section without the application of TMab-6 An antibody absorption test with synthetic neutralization peptide of TERT furthervalidated 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 antibodymt mutation Scale bar = 40 120583m

recognized human TERT protein and that it could beapplied for immunohistochemistry on the human gliomatissue 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 TERThotspot mutations as well as upregulated TERT transcripts[5 33]

33 Expression of TERT in Human Glioma Tissue After con-firming the specificity of a newly developed anti-TERTmon-oclonal antibodyTMab-6we performed immunohistochem-ical staining on sections of nonneoplastic adult human cere-bral tissues and all glioma samples including IDH-mutatedastrocytoma oligodendroglioma IDH-wildtype GBM andIDH-wildtype diffuse astrocytoma and examined the staining


Astro (AA)(TERT wt)

Oligo (AO)(TERT mt C228T)

IDHwt GBM(TERT mt C228T)

HampE TERT

IDHwt (AA)(TERT wt)

IDHwt (AOA)(TERT mt C228T)

(a)

Control (nonneoplastic tissue)

Case Astro_4 (AA)

Case Oligo_10 (AO)

Case GBM_5 (IDHwt GBM)

TERT WT

TERT mt C228T

TERT WT

TERT WT

(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 GBMand 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-mutatedAO and TERT-mutated GBM AA anaplastic astrocytoma AO anaplastic oligodendroglioma AOA anaplastic oligoastrocytoma GBMIDH-wildtype glioblastoma mt mutation wtWT wildtype Scale bar = 40 120583m

patterns of TERT protein in them Immunohistochemicalobservations using TMab-6 demonstrated strong staining inthe nucleus and some cytoplasmic staining of the gliomacells (Figure 3(a)) and TERT immunoreactivity was notobserved in the normal tissue adjacent to the tumor tissueSurprisingly glioma cells in all the cases showed nuclearimmunostaining for TERT to various degrees compared withthe cells in nonneoplastic tissues (Figure 3(a)) althoughsome intratumoral heterogeneity of the nuclear staining was

observed Some reactive astrocytes in cerebral parenchymafrom the cases with hippocampal sclerosis and corticaldysplasia showed nonspecific weak cytoplasmic staining(Supplementary Figure 2) Interestingly endothelial cells ofthe tumor vessels also displayed nuclear immunostainingin both TERT-mutated and wildtype tumors whereas vas-cular endothelial cells in the nonneoplastic tissues did notshow any specific TERT immunoreactivity in their nuclei(Figure 3(b))


34 Semiquantitative Evaluation of TERT Expression Asso-ciated with TERT Promoter Mutations in Human GliomasHaving confirmed the findings that all the glioma cellswe analyzed showed nuclear immunostaining of TERT tovarious degrees we further examined the association ofthe TERT promoter mutations with TERT immunoreactivityin our glioma samples Unexpectedly immunohistochem-istry using TMab-6 demonstrated that immunoreactivity forTERT did not correspond to the mutational status of theTERT promoter region across glioma samples (Figure 4(a))Although the staining was weak nuclear immunoreactivityfor TERT was also confirmed in both TERT-wildtype andmutated gliomas with the commercially available antibodyfor TERT (sc-7215) (Supplementary Figure 1) In additionto the hotspot mutations in the TERT promoter (C228Tand C250T) the presence of a common SNP in the TERTpromoter (T349C) was not associated with TERT immunore-activity (Supplementary Table 1) TERT immunohistochem-istry was not capable of identifying the differences betweenTERT-mutated gliomas and TERT-nonmutant gliomas (Fig-ure 4(a)) The scores might be relevant to tumor gradingand aggressiveness represented by IDH-wildtype GBMs andIDH-wildtype diffuse astrocytomas (Figure 4(a)) both ofwhich behave aggressively with a dismal prognosis [1 4 1421] Finally the relationship between the TERT promotermutations the expression levels of TERT protein and tumorentity was further verified by Western blotting (Figure 4(b))and the level of TERT mRNA was not parallel with its muta-tional status across all the glioma samples (data not shown)

4 Discussion

The mechanism of TERT upregulation in cancer has largelyremained unknown to date Recurrent mutations at twohotspot regions (C228T and C250T) in the TERT promoterhave been reported in various types of cancers [2 3 5]and a recent report outlined the mechanism that TERTpromoter mutations reactivate TERT expression [34] Indiffuse gliomas the sole mechanism to upregulate TERTmRNA is considered to be two hotspot mutations in theTERT promoter region However the level of TERT tran-scripts varies widely in expression among tumors includinggliomas [5 7] skin cancers [2 3 35] thyroid cancers [7]hepatocellular carcinomas [36] bladder cancers [37] andmalignant lymphomas [38] The levels of TERT mRNA tendto be increased in TERT-mutated tumors in comparisonwith TERT-wildtype tumors but it is not necessarily thecase for the individual tumors indicating that mutationsin the TERT promoter alone could not explain the widevariety in the expression of TERT transcripts Further tothe best of our knowledge only one report has so farinvestigated the expression of TERT protein in gliomas andany difference was not detected between TERT-mutated andTERT-wildtype GBMs [7]

In the present study we compared the expression of TERTprotein with its mutational status in adult gliomas In linewith the previous studies TERT expression was confirmedin the nuclei of oligodendroglial tumors and IDH-wildtypeGBM which possess either of two hotspot mutations in the

promoter regions of TERT Surprisingly however even IDH-mutant astrocytic tumors (DAAA) without known hotspotmutations in the TERT promoter displayed certain amountof TERT immunoreactivity in their nuclei Additionally qRT-PCR study did not demonstrate any significant correlationbetween TERT promoter mutations and the level of TERTmRNA expression Considering our findings that immuno-precipitationwith TMab-6 did demonstrate the same blottingbands as the commercially available antibody (10E9-2sc-7215) and TMab-6 and sc-7215 showed the similar immunos-taining pattern in the tumor TERT immunoreactivity issurely increased in tumor tissue in comparison with nor-mal tissue although the possibility of any unknown cross-reactivity cannot be completely ruled out The finding thatthe TERT immunoreactivity in endothelial as well as tumorcells is strictly limited to the neoplastic tissue further supportsthe idea that TMab-6 detects TERT protein closely boundedto 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 proteincould be regulated by themechanism other than its promotermutations including via epigenetic factors In addition toreported hotspot mutations certain SNPs in the TERTpromoter region (T349C) were reported to suppress TERTtranscripts [32] but we could not confirm any associationof SNPs with TERT protein expression further supportingour hypothesis that TERT expression could be epigeneticallyregulated

There could be several mechanisms to increase theexpression of TERT protein in TERT-wildtype tumors It hasbeen reported that DNA hypermethylation of the TERT pro-moter is associatedwithTERTupregulation in pediatric braintumors [8] and other tumors [39] and histone acetylationat the TERT promoter regions may also be relevant to itsexpression [9] IDH-mutant diffuse astrocytic tumors oftendemonstrate the glioma-CpG island methylator phenotype(G-CIMP) [40 41] and the nuclear TERT staining in diffuseastrocytomas might be epigenetically caused by hypermethy-lation of the TERT promoter [10] Additionally a numberof human tumors maintain their telomeres by a telomerase-independent mechanism termed alternative lengthening oftelomeres (ALT) caused by such abnormalities as loss ofATRX [42 43] and this might somehow affect the expressionlevel of TERT

In addition to its applicability to the molecular diagnos-tics of gliomas TERT could be a good therapeutic targetagainst diffuse gliomas which are a potentially malignantincurable brain tumor in human A novel TERT-targetingtherapy [44 45] would be expected to specifically targetIDH-wildtype GBM including primary brain tumor initi-ating cells and 1p19q-codeleted oligodendroglioma whichhave characteristically possessed hotspot mutations in theTERT promoter region [5 33] Our unexpected findingssuggest that a specific TERT-targeting therapy might be apromising therapeutic strategy against all types of gliomas bydirectly targeting tumor cells with an indirect antiangiogeniceffect on tumor vasculature like the antiangiogenic agent


3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a

TERT immunoreactivity

TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6

TERT protein expression

Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH

Astro Oligo GBM IDHwt DA

1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)

Figure 4 Association of TERT promoter mutations and its immunoreactivity in human gliomas (a) Immunostaining of TERT by TMab-6demonstrated that TERT immunoreactivity did not correspond to the mutational status of the TERT promoter region across glioma samplesSemiquantitative analysis of the TERT-immunostaining scoring the staining intensities as 1+ (a few weakly positive cells) 2+ (positive cells lt50) and 3+ (positive cellsge 50) Blue dots representTERT-wildtype cases and red denotesTERT-mutated cases119875 values for nonneoplastictissue versus each glioma entity (b) An analysis on the relationship of the TERT promoter mutations the expression level of TERT proteinand each tumor entity by Western blotting with the most widely used commercially available TERT antibody (sc-7215) A bar graph showedthe quantification of TERT expression in each case normalized by a loading control GAPDH expression lowastNonspecific bands LE longexposure AA anaplastic astrocytoma AO anaplastic oligodendroglioma AOA anaplastic oligoastrocytoma CD cortical dysplasia DAdiffuse astrocytoma GBM IDH-wildtype glioblastoma HS hippocampal sclerosis OA oligoastrocytoma OL oligodendroglioma IDHwtIDH-wildtype diffuse astrocytoma wtWT wildtype Scale bar = 40 120583m

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

biology for the recommendation of treatment based on TERTimmunohistochemistry

The limitation of our study is that we have not directlymeasured the telomerase activity itself Indeed some reports


demonstrated that TERT expression and its biological activityare not necessarily parallel [12] Further there could be thepossibility that this novel antibody is picking up baselineTERT protein that may not be associated with increasedtelomerase efficiency or activity It would be thus importantto further validatespecify the antibody and analyze therelationship between histological TERT immunoreactivityand its enzymatic activity as a next step Future studiesare also necessary to unravel the contributing factors inupregulating the protein expression of TERT to exam-ine if TERT protein expression could be the biomarkerfor the applicability of TERT-targeting therapeutics in thefuture

Together we developed a novel TERT-specific antibodyand it is sensitively and specifically applicable to immunohis-tochemistry for TERT in human glioma tissue Immunohis-tochemistry with this TERT-specific antibody could not playthe diagnostic surrogate for TERT promoter mutations butwe could find an unexpected increase in TERT immunore-activity across all types of gliomas and tumor vasculatureThis study is the first extensive analysis on the expressionof TERT protein in human glioma tissue and suggests thatTERT expression could be regulated by various mechanismsincluding epigenetic modifications Future studies with themeasurement of TERT activity could be useful for facilitatingour understanding of telomere biology in cancer includinggliomas

Disclosure

A part of this work was presented at 11th European Congressof Neuropathology (July 6ndash9 2016 Bordeaux France) and13th Asian Society for Neuro-Oncology (September 11ndash142016 Sydney Australia)

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

K Masui and T Komori contributed equally to the work

Acknowledgments

The authors would like to thank Mr Mizuho KaritaMr Takashi Sakayori (Tokyo Womenrsquos Medical Univer-sity) and Dr Mami Yasukawa (Division of Cancer StemCell National Cancer Center Research Institute) for theirexcellent 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-in-Aid from the Tokyo Biochemical Research Foundationand JSPS KAKENHI Grant nos 15K19067 and 17K15672(Kenta Masui)

Supplementary Materials

Supplementary Table 1 Detailed information of the casesSupplementary Table 2 Primer informationSupplementary Figure 1 Immunostaining of TERT proteinin human gliomas with TMab-6 and sc-7215 We immunos-tained TERT-wildtype and mutant gliomas with our newlydeveloped TMab-6 and the most widely used commer-cially available sc-7215 Both antibodies detected nuclearimmunoreactivity of TERT in TERT-wildtype and mutatedgliomas although the staining intensities were variable AAanaplastic astrocytoma AO anaplastic oligodendrogliomaScale bar = 40 120583mSupplementary Figure 2 Immunostaining of TERT proteinin human nonneoplastic brain tissueWe immunostained thecases of TERT-wildtype hippocampal sclerosis and corticaldysplasia with our newly developed TMab-6 (05 120583gmL)Some reactive astrocytes showed nonspecific weak cytoplas-mic staining although any nuclear staining of TERT was notobserved in them

References

[1] D J Brat R G Verhaak K D Aldape et al et al ldquoCom-prehensive integrative genomic analysis of diffuse lower-gradegliomasrdquo The New England Journal of Medicine vol 372 pp2481ndash2498 2015

[2] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[3] F W Huang E Hodis M J Xu G V Kryukov L Chin andL A Garraway ldquoHighly recurrent TERT promoter mutationsin human melanomardquo Science vol 339 no 6122 pp 957ndash9592013

[4] J E Eckel-Passow D H Lachance A M Molinaro et al et alldquoGlioma Groups Based on 1p19q IDH and TERT PromoterMutations in Tumorsrdquo The New England Journal of Medicinevol 372 pp 2499ndash2508 2015

[5] H Arita YNarita S Fukushima et al ldquoUpregulatingmutationsin the TERT promoter commonly occur in adult malignantgliomas and are strongly associated with total 1p19q lossrdquo ActaNeuropathologica vol 126 no 2 pp 267ndash276 2013

[6] H Arita Y Narita H Takami et al ldquoTERT promotermutationsrather than methylation are the main mechanism for TERTupregulation in adult gliomasrdquo Acta Neuropathologica vol 126no 6 pp 939ndash941 2013

[7] J Vinagre A Almeida H Populo et al ldquoFrequency of TERTpromoter mutations in human cancersrdquo Nature Communica-tions vol 4 article 2185 2013

[8] P Castelo-Branco S Choufani S Mack et al ldquoMethylation ofthe TERT promoter and risk stratification of childhood braintumours An integrative genomic and molecular studyrdquo TheLancet Oncology vol 14 no 6 pp 534ndash542 2013

[9] B Zhang J Chen A S L Cheng and B C B Ko ldquoDepletion ofsirtuin 1 (SIRT1) leads to epigenetic modifications of telomerase(TERT) gene in hepatocellular carcinoma cellsrdquo PLoS ONE vol9 no 1 Article ID e84931 2014


[10] S Ohba J Mukherjee T-C Johannessen et al ldquoMutant IDH1expression drives TERT promoter reactivation as part of thecellular transformation processrdquo Cancer Research vol 76 no22 pp 6680ndash6689 2016

[11] N W Kim M A Piatyszek K R Prowse et al ldquoSpecificassociation of human telomerase activity with immortal cellsand cancerrdquo Science vol 266 no 5193 pp 2011ndash2015 1994

[12] M Ceccarelli F P Barthel T M Malta et al ldquoMolecularprofiling reveals biologically discrete subsets and pathways ofprogression in diffuse gliomardquo Cell vol 164 pp 550ndash563 2016

[13] T Komori ldquoUpdated 2016 WHO classification of tumors ofthe CNS turning the corner where molecule meets pathologyrdquoBrain Tumor Pathology vol 34 no 4 pp 139-140 2017

[14] D N Louis A Perry G Reifenberger et al ldquoThe 2016 WorldHealth Organization Classification of Tumors of the CentralNervous System a summaryrdquo Acta Neuropathologica vol 131no 6 pp 803ndash820 2016

[15] A Perry ldquoWHOrsquos arrived in 2016 An updated weather forecastfor integrated brain tumor diagnosisrdquo Brain Tumor Pathologyvol 33 no 3 pp 157ndash160 2016

[16] D N Louis A Perry P Burger et al ldquoInternational societyof neuropathology-haarlem consensus guidelines for nervoussystem tumor classification and gradingrdquo Brain Pathology vol24 no 5 pp 429ndash435 2014

[17] K Masui P S Mischel and G Reifenberger ldquoMolecularclassification of gliomasrdquo Handbook of Clinical Neurology vol134 pp 97ndash120 2016

[18] H Takami A Yoshida S Fukushima et al ldquoRevisiting TP53mutations and immunohistochemistry - A comparative studyin 157 diffuse gliomasrdquo Brain Pathology vol 25 no 3 pp 256ndash265 2015

[19] X-Y Liu N Gerges A Korshunov et al ldquoFrequent ATRXmutations and loss of expression in adult diffuse astrocytictumors carrying IDH1IDH2 and TP53 mutationsrdquo Acta Neu-ropathologica vol 124 no 5 pp 615ndash625 2012

[20] D E Reuss F Sahm D Schrimpf et al ldquoATRX and IDH1-R132H immunohistochemistry with subsequent copy numberanalysis and IDH sequencing as a basis for an ldquointegratedrdquodiagnostic approach for adult astrocytoma oligodendrogliomaand glioblastomardquo Acta Neuropathologica vol 129 no 1 pp133ndash146 2015

[21] K Aldape G Zadeh S Mansouri G Reifenberger and A vonDeimling ldquoGlioblastoma pathology molecular mechanismsand markersrdquo Acta Neuropathologica vol 129 no 6 pp 829ndash848 2015

[22] S Ogasawara Y Fujii M K Kaneko et al ldquoEstablishment ofAnti-Human ATRX Monoclonal Antibody AMab-6rdquo Mono-clonal Antibodies in Immunodiagnosis and Immunotherapy vol35 no 5 pp 254ndash258 2016

[23] M J Van Den Bent A F Carpentier A A Brandes et alldquoAdjuvant procarbazine lomustine and vincristine improvesprogression-free survival but not overall survival in newly diag-nosed anaplastic oligodendrogliomas and oligoastrocytomas Arandomized European Organisation for Research and Treat-ment of Cancer phase III trialrdquo Journal of Clinical Oncology vol24 no 18 pp 2715ndash2722 2006

[24] H Arita Y Narita Y Matsushita et al ldquoDevelopment of arobust and sensitive pyrosequencing assay for the detection ofIDH12 mutations in gliomasrdquo Brain Tumor Pathology vol 32no 1 pp 22ndash30 2014

[25] H Arita Y Narita A Yoshida N Hashimoto T YoshimineandK Ichimura ldquoIDH12mutation detection in gliomasrdquoBrainTumor Pathology vol 32 no 2 pp 79ndash89 2015

[26] Y Fujii M K Kaneko and Y Kato ldquoMAP Tag ANovel TaggingSystem for Protein Purification and Detectionrdquo MonoclonalAntibodies in Immunodiagnosis and Immunotherapy vol 35 no6 pp 293ndash299 2016

[27] Y Fujii M KanekoM Neyazaki T Nogi Y Kato and J TakagildquoPA tag A versatile protein tagging system using a super highaffinity antibody against a dodecapeptide derived from humanpodoplaninrdquo Protein Expression and Purification vol 95 pp240ndash247 2014

[28] Y Maida M Yasukawa N Okamoto et al ldquoInvolvement oftelomerase reverse transcriptase in heterochromatin mainte-nancerdquoMolecular and Cellular Biology vol 34 no 9 pp 1576ndash1593 2014

[29] Y Maid M Yasukaw and K Masutomi ldquoDe novo RNA synthe-sis by RNAdependent RNA polymerase activity of telomerasereverse transcriptaserdquo Molecular and Cellular Biology vol 36no 8 pp 1248ndash1259 2016

[30] K Masui K Tanaka D Akhavan et al ldquoMTOR complex 2controls glycolytic metabolism in glioblastoma through FoxOacetylation and upregulation of c-MycrdquoCell Metabolism vol 18no 5 pp 726ndash739 2013

[31] K Masui K Tanaka S Ikegami et al ldquoGlucose-dependentacetylation of Rictor promotes targeted cancer therapy resis-tancerdquo Proceedings of the National Acadamy of Sciences ofthe United States of America vol 112 no 30 pp 9406ndash94112015

[32] C-K Park S-H Lee J Y Kim et al ldquoExpression level of hTERTis regulated by somaticmutation and common single nucleotidepolymorphism at promoter region in glioblastomardquoOncotarget vol 5 no 10 pp 3399ndash3407 2014

[33] T M Johanns Y Fu D K Kobayashi et al ldquoHigh incidenceof TERT mutation in brain tumor cell linesrdquo Brain TumorPathology vol 33 no 3 pp 222ndash227 2016

[34] R J A Bell H T Rube A Kreig et al ldquoThe transcriptionfactor GABP selectively binds and activates the mutant TERTpromoter in cancerrdquo Science vol 348 no 6238 pp 1036ndash10392015

[35] H Xie T Liu N Wang et al ldquoTERT promoter mutations andgene amplification Promoting TERT expression in merkel cellcarcinomardquo Oncotarget vol 5 no 20 pp 10048ndash10057 2014

[36] J C Nault M Mallet C Pilati et al ldquoHigh frequency oftelomerase reverse-transcriptase promoter somatic mutationsin hepatocellular carcinoma and preneoplastic lesionsrdquo NatureCommunications vol 4 p 2218 2013

[37] S Borah L Xi A J Zaug et al ldquoTERT promoter mutations andtelomerase reactivation in urothelial cancerrdquo Science vol 347no 6225 pp 1006ndash1010 2015

[38] J Panero R M Alves-Paiva A Roisman et al ldquoAcquired TERTpromoter mutations stimulate TERT transcription in mantlecell lymphomardquo American Journal of Hematology vol 91 no5 pp 481ndash485 2016

[39] I Guilleret P Yan F Grange R Braunschweig F T Bosmanand J Benhattar ldquoHypermethylation of the human telomerasecatalytic subunit (hTERT) gene correlates with telomeraseactivityrdquo International Journal of Cancer vol 101 no 4 pp 335ndash341 2002


[40] H Noushmehr D J Weisenberger K Diefes et al ldquoIdentifi-cation of a CpG island methylator phenotype that defines adistinct subgroup of gliomardquo Cancer Cell vol 17 no 5 pp 510ndash522 2010

[41] KMasuiW K Cavenee and P SMischel ldquoCancermetabolismas a central driving force of glioma pathogenesisrdquo Brain TumorPathology vol 33 no 3 pp 161ndash168 2016

[42] C M Heaphy R F deWilde Y Jiao et al ldquoAltered telomeres intumors with ATRX and DAXXmutationsrdquo Science vol 333 no6041 article 425 2011

[43] J Schwartzentruber A Korshunov X Y Liu et al ldquoDrivermutations in histone H33 and chromatin remodelling genes inpaediatric glioblastomardquo Nature vol 482 pp 226ndash231 2012

[44] Y Maida and K Masutomi ldquoTelomerase reverse transcriptasemoonlights Therapeutic targets beyond telomeraserdquo CancerScience vol 106 no 11 pp 1486ndash1492 2015

[45] S Yamaguchi Y Maida M Yasukawa T Kato M Yoshidaand KMasutomi ldquoEribulin mesylate targets human telomerasereverse transcriptase in ovarian cancer cellsrdquo PLoS ONE vol 9no 11 Article ID e112438 2014

[46] O L Chinot W Wick W Mason et al ldquoBevacizumabplus radiotherapy-temozolomide for newly diagnosed glioblas-tomardquoThe New England Journal of Medicine vol 370 no 8 pp709ndash722 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


in various cancers including gliomas [5 7] and there maybe other potential mechanisms to upregulate TERT mRNAin addition to its promoter mutations Indeed in other braintumors and systemic cancers epigenetic shifts in the TERTpromoter regions including DNA methylation and histonemodifications were reported to regulate TERT transcriptsand reactivation [8ndash10] necessitating a rethinking of themechanism to upregulate the expression of TERT

As an essential component of telomerase TERT addstelomeric repeats to chromosomal ends using telomeraseRNA component (TERC) as a template and maintainstelomere length Telomerase activity had been considered tobe maintained in most types of the cancer cells enablingthem to proliferate and survive indefinitely [11] However arecent report demonstrated that telomere length is positivelycorrelated with mutations in the alpha thalassemiamentalretardation syndrome X-linked gene (ATRX) but not theTERT promoter suggesting that TERT upregulation itselfmay be essential for glioma biology irrespective of telomerelength [12] It would be thus critical to examine the expressionlevel of TERT protein in diffuse gliomas to clarify the role ofTERT in their biology in addition to its mRNA level as wellas mutational status but few reports have so far validatedthe expression of TERT protein in human glioma tissue[7] Sensitive and specific methods are strongly desired toexamine TERT protein expression for the future analyses onthe TERT biology in diffuse gliomas

In this study we developed a TERT-specific antibodyapplicable to human tissue and examined the expression ofTERT in a series of glioma samples Although the practicaldiagnostic utility of the antibody to detect the TERT-mutatedtumors was expected TERT immunohistochemistry wasnot capable of identifying the differences between TERT-mutated gliomas and TERT-nonmutant gliomas Surpris-ingly however an increase in TERT protein expressionwas detected across all types of gliomas including tumorswith wildtype TERT and the level of TERT expressionwas different even among the TERT-mutant gliomas suchas oligodendrogliomas and GBMs Our study is the firstthorough analysis on the expression of TERT protein using aspecific TERT monoclonal antibody in human glioma tissuesuggesting that TERT protein expressionmay be regulated byseveral mechanisms in addition to its promoter mutation

2 Materials and Methods

21 Tissue Collection A total of 41 human glioma tissuesand 4 nonneoplastic adult human cerebral tissues fromfour epileptic patients were obtained at surgery from TokyoWomenrsquos Medical University Hospital and Tokyo Metropoli-tan Neurological Hospital The tumor samples included 11grade II and III astrocytic tumors with IDH mutations[diffuse astrocytoma (DA) and anaplastic astrocytoma (AA)]10 grade II and III oligodendroglial tumors 11 grade IVIDH-wildtype GBMs and 9 IDH-wildtype diffuse astrocy-tomas including tumors histologically diagnosed as diffuseastrocytoma anaplastic astrocytoma oligoastrocytoma andanaplastic oligoastrocytoma which did not show IDH muta-tions p53 immunopositivity and the loss of ATRX expression

(wtIDHp53-ATRX+) Clinical information of the cases wascollected from the medical records All specimens werefixed in 10 buffered formalin and embedded into paraffinsections Sections were routinely stained with hematoxylinand eosin (HampE) and histopathological diagnosesweremadeaccording to the World Health Organization (WHO) 2016classification of tumors of the nervous system [13ndash15] andInternational Society of Neuropathology-Haarlem consensusguidelines [16]

All tumor and control brain samples were subjected tohistological diagnosis followed by molecular genetics screen-ing for a more precisemolecular diagnosis (Figure 1(a)) [17]Tumors in the astrocytoma group (DAAA) were charac-terized by detectable immunoreactivity for mutated IDH1(R132H) and p53 [18] and the loss of immunoreactivity forATRX [19 20] All tumors in the oligodendroglioma groupdemonstrated mutated IDH1 (R132H) and 1p19q-codeletionGBM tumors did not possess mutations in IDH correspond-ing to ldquoGBM IDH-wildtyperdquo [14 21] The tumors in theIDH-wildtype diffuse astrocytoma group did not show anymolecular genetic aberrations shown above The study wasapproved by the institutional review boards and the detailedinformation of the cases was provided in SupplementaryTable 1

22 DNA Extraction and Sanger Sequencing Genomic DNAwas extracted from formalin-fixed paraffin-embedded(FFPE) tissue of the solid tumor regions confirmed on HampEsections using QIAamp DNA FFPE Tissue Kit (QIAGENVenlo The Netherlands) followed by the purification withNucleoSpin gDNA Clean-up Kit (MACHEREY-NAGELDusseldorf Germany) according to the manufacturerrsquosinstructions Sequences of the primer sets for polymerasechain reaction (PCR) were provided in SupplementaryTable 2 PCR was performed using AmpliTaq Gold 360 DNAPolymerase and GC enhancer (Thermo Fisher ScientificWaltham MA) Sequencing was carried out by EurofinsGenomics (Tokyo Japan) with each forward PCR primer asa sequencing primer

23 Molecular Diagnostics The status of IDH1 (R132H) p53and ATRXwas analyzed by immunohistochemistry using theautomated immunostaining processor Histostainer (NichireiBiosciences Tokyo Japan)The antibodies used include anti-IDH1 (R132H) (DIA-H09 Dianova Hamburg Germany)anti-p53 (DO-7 Nichirei) and anti-ATRX (HPA001906Sigma-Aldrich St Louis MO) We judged the tumor as p53-positive when gt50 of the tumor nuclei displayed the strongimmunoreactivity of p53 which could be corroborated by theloss of immunoreactivity for ATRX (inactivating mutation)and 1p19q-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 19q13probes (Vysis Abbott Molecular Abbott Park IL) and signalratios were calculated according to the previous report [23]Two reported hot spot mutations (C228T and C250T) in aTERT promoter region were analyzed by Sanger sequencingThe hotspot mutations at codon 132 of IDH1 and codon 172of IDH2 were also screened by Sanger sequencing if the cases


AA

AO

GBM


AOA (IDHwt)

(a)


Astro_1 (AA)

TERT wildtype(C228)

GBM_1 (IDHwt GBM)



TERT wildtype(C250)


(b)

0

20

40

60

80

100

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

-wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


C228TC250T

Mut

atio

n fre

quen

cy (

)

(c)

TERT SNP(T349C)


TERT SNP(T349)





















3 Results





Protein L Protein A

Prei

mm

une

Irre

leva

nt Ig

M

TMab

-6

10

E9-2

nt

150

100908070

60

50

40

30

20

(a)


(TMab-6)

WithTERT primary Ab



WithTERT primary Ab


(Ab peptide = 1 1)

WithTERT primary Ab


(b)


G


C C CCCC TT

(c)





Astro (AA)(TERT wt)



HampE TERT

IDHwt (AA)(TERT wt)


(a)


Case Astro_4 (AA)

Case Oligo_10 (AO)


TERT WT

TERT mt C228T

TERT WT

TERT WT

(b)






4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















AA

AO

GBM


AOA (IDHwt)

(a)


Astro_1 (AA)

TERT wildtype(C228)

GBM_1 (IDHwt GBM)



TERT wildtype(C250)


(b)

0

20

40

60

80

100

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

-wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


C228TC250T

Mut

atio

n fre

quen

cy (

)

(c)

TERT SNP(T349C)


TERT SNP(T349)





















3 Results





Protein L Protein A

Prei

mm

une

Irre

leva

nt Ig

M

TMab

-6

10

E9-2

nt

150

100908070

60

50

40

30

20

(a)


(TMab-6)

WithTERT primary Ab



WithTERT primary Ab


(Ab peptide = 1 1)

WithTERT primary Ab


(b)


G


C C CCCC TT

(c)





Astro (AA)(TERT wt)



HampE TERT

IDHwt (AA)(TERT wt)


(a)


Case Astro_4 (AA)

Case Oligo_10 (AO)


TERT WT

TERT mt C228T

TERT WT

TERT WT

(b)






4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





































3 Results





Protein L Protein A

Prei

mm

une

Irre

leva

nt Ig

M

TMab

-6

10

E9-2

nt

150

100908070

60

50

40

30

20

(a)


(TMab-6)

WithTERT primary Ab



WithTERT primary Ab


(Ab peptide = 1 1)

WithTERT primary Ab


(b)


G


C C CCCC TT

(c)





Astro (AA)(TERT wt)



HampE TERT

IDHwt (AA)(TERT wt)


(a)


Case Astro_4 (AA)

Case Oligo_10 (AO)


TERT WT

TERT mt C228T

TERT WT

TERT WT

(b)






4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























3 Results





Protein L Protein A

Prei

mm

une

Irre

leva

nt Ig

M

TMab

-6

10

E9-2

nt

150

100908070

60

50

40

30

20

(a)


(TMab-6)

WithTERT primary Ab



WithTERT primary Ab


(Ab peptide = 1 1)

WithTERT primary Ab


(b)


G


C C CCCC TT

(c)





Astro (AA)(TERT wt)



HampE TERT

IDHwt (AA)(TERT wt)


(a)


Case Astro_4 (AA)

Case Oligo_10 (AO)


TERT WT

TERT mt C228T

TERT WT

TERT WT

(b)






4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Protein L Protein A

Prei

mm

une

Irre

leva

nt Ig

M

TMab

-6

10

E9-2

nt

150

100908070

60

50

40

30

20

(a)


(TMab-6)

WithTERT primary Ab



WithTERT primary Ab


(Ab peptide = 1 1)

WithTERT primary Ab


(b)


G


C C CCCC TT

(c)





Astro (AA)(TERT wt)



HampE TERT

IDHwt (AA)(TERT wt)


(a)


Case Astro_4 (AA)

Case Oligo_10 (AO)


TERT WT

TERT mt C228T

TERT WT

TERT WT

(b)






4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Astro (AA)(TERT wt)



HampE TERT

IDHwt (AA)(TERT wt)


(a)


Case Astro_4 (AA)

Case Oligo_10 (AO)


TERT WT

TERT mt C228T

TERT WT

TERT WT

(b)






4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















4 Discussion








3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















3 (OL)

C250T

1 (DA)

WT

5 (GBM)

C250T

7 (AOA)

WT

6 (GBM)

C250T

4 (AO)

C250T

2 (AA)

WT

8 (OA)

C250T

0

1

2

3

4

Non

neop

lasti

c(H

S C

D)

Astr

ocyt

ic w

ithID

H-m

utan

t(D

A A

A)

Olig

oden

drog

lial

(OL

AO

)

IDH

wild

type

GBM

IDH

-wild

type

diffu

seas

trocy

tom

a


TERT

IHC

scor

e

1+

2+ 3+

Score p = 007p = 009 p = p = 2

(a)

0

2

4

6

8

10

A1

A2

OL

3

OL

4

GB

5

GB

6


Relat

ive e

xpre

ssio

n

239 134

IDH

wt

7

IDH

wt

8

52

76102150225

GAPDH


1 2 3 4 5 6 7 8

TERT(LE)

38(kDa)

TERT(120 kDa)

lowast

lowast

(b)








Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Disclosure






Acknowledgments




References





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Elevated TERT Expression in TERT-Wildtype Adult Diffuse ...downloads.hindawi.com/journals/bmri/2018/7945845.pdf · ResearchArticle Elevated TERT Expression in TERT-Wildtype Adult