Neuron-glial antigen 2 overexpression in hepatocellular carcinoma predicts poor prognosis

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v21.i World J Gstroenterol 215 June 7; 21(21): ISSN (print) ISSN (online) 215 Bishideng Publishing Group Inc. All rights reserved. Retrospective Study ORIGINAL ARTICLE Neuron-glil ntigen 2 overexpression in heptocellulr crcinom predicts poor prognosis Le-Le Lu, Jing Sun, Jie-Jun Li, Yn Jing, Lin-Hu Bi, Lei-D Zhng Le-Le Lu, Jing Sun, Jie-Jun Li, Yn Jing, Lin-Hu Bi, Lei-D Zhng, Deprtment of Heptobiliry Surgery, Southwest Hospitl, Third Militry Medicl University, Chongqing 438, Chin Author contributions: Lu LL nd Sun J contributed eqully to this work; Lu LL nd Sun J performed the mjority of experiments nd wrote the mnuscript; Li JJ nd Jing Y provided vitl regents nd nlyticl tools nd were lso involved in editing the mnuscript; Bi LH nd Zhng LD coordinted nd provided the collection of ll the humn mteril in ddition to designing the study nd providing finncil support for this work. Supported by Ntionl Nturl Science Foundtion of Chin, No nd No ; Heptobiliry Surgery Deprtment of the Southwest Hospitl (Chongqing, Chin); nd Third Militry Medicl University (Chongqing, Chin). Ethics pprovl: All tissue smples were collected from heptocellulr crcinom ptients who underwent heptectomy t the Deprtment of Heptobiliry Surgery between Jnury nd December 27, ccording to protocols pproved by the Medicl Ethics Committee of the Southwest Hospitl nd the First Affilited Hospitl of the Third Militry Medicl University. Informed consent: All ptients provided written informed consent to use tumor tissue for clinicl reserch. Conflict-of-interest: No conflict of interest exits in the submission of this mnuscript, nd the mnuscript is pproved by ll uthors for publiction. Dt shring: Prticipnts gve informed consent for dt shring. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4./ Correspondence to: Lei-D Zhng, MD, PhD, Deprtment of Heptobiliry Surgery, Southwest Hospitl, Third Militry Medicl University, Shpingb No. 3 Gotnyn Street, Chongqing 438, Chin @qq.com Telephone: Fx: Received: September 1, 214 Peer-review strted: September 5, 214 First decision: October 14, 214 Revised: December 3, 214 Accepted: Jnury 16, 215 Article in press: Jnury 16, 215 Published online: June 7, 215 Abstrct AIM: To investigte whether neuron-glil ntigen 2 (NG2) could be n effective prognostic mrker in heptocellulr crcinom (HCC). METHODS: NG2 expression ws semi-quntittively scored from the immunohistochemistry (IHC) dt bsed on the number of positive cells nd the stining intensity. A totl of 132 HCC specimens nd 96 djcent noncncerous tissue smples were nlyzed by IHC for NG2 protein expression. To confirm the NG2 expression levels observed by IHC, we mesured NG2 expression in 3 rndomly selected tumor nd djcent noncncerous tissue smples by quntittive rel-time polymerse chin rection nd Western blot. The correltions between NG2 protein expression nd the clinicopthologicl fetures of HCC ptients were nlyzed using the χ 2 test. To ssess the prognostic vlue of NG2 for HCC, the ssocition between NG2 expression nd survivl ws nlyzed using the Kpln- Meier method with the log-rnk test. To further evlute the prognostic vlue of NG2 expression, Cox multivrite proportionl hzrds regression nlysis ws performed with ll the vribles to derive risk estimtes relted to disese-free nd overll survivl nd to control for confounders June 7, 215 Volume 21 Issue 21

2 Lu LL et l. NG2 expression in heptocellulr crcinom RESULTS: High NG2 expression ws observed in significntly more primry tumor smples (63.6%; 84/132) compred with the djcent noncncerous tissue smples (28.1%; 27/96) (P <.1). Moreover, high NG2 protein expression ws closely ssocited with tumor differentition (χ 2 = 9.436, P =.89), recurrence (χ 2 = 5.769, P =.163), tumor-nodemetstsis (TNM) stge (χ 2 = 8.976, P =.27), nd invsion (χ 2 = 5.476, P =.193). However, no significnt reltionship ws observed between NG2 protein expression in HCC nd other prmeters, such s ge, sex, tumor size, serum lph fetoprotein (AFP), tumor number, or tumor cpsule. The log-rnk test indicted significnt difference in the overll survivl of HCC ptients with high NG2 expression compred with those with low NG2 expression (29.2% vs 9.5%, P <.1). Moreover, NG2 expression in HCC tissue significntly correlted with disese-free survivl (15.2% vs 6.7%, P <.1). Multivrite nlysis showed tht NG2 expression (HR = 2.35, P =.2), serum AFP (HR = 1.93, P =.3), TNM stge (HR = 2.39, P =.1), nd portl vein invsion (HR = 1.938, P =.2) were independent prognostic indictors for OS in HCC ptients. Furthermore, NG2 expression (HR = 1.974, P =.3), serum AFP (HR = 1.767, P =.8), TNM stge (HR = 2.78, P =.1), tumor cpsule (HR =.652, P =.45), nd portl vein invsion (HR = 1.941, P =.2) were independent prognostic indictors for DFS in HCC ptients. CONCLUSION: The up-regultion of NG2 is ssocited with poor prognosis in HCC. Therefore, NG2 could be useful s n dditionl prognostic mrker to increse the resolution of trditionl pproches. Key words: Neuron-glil ntigen 2; Heptocellulr crcinom; Survivl nlysis; Poor prognosis; Prognostic mrker The Author(s) 215. Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: To help predict which ptients hve n unfvorble prognosis, it is criticl to identify dependble prognostic biomrker tht correltes with heptocellulr crcinom (HCC) progression, invsion, metstsis nd recurrence. Despite recent evidence showing tht neuron-glil ntigen 2 (NG2) is expressed on the surfce of differentited mlignnt cells, progenitor cells, nd cncer stem cells in vrious tumors nd promotes the growth nd metstsis of melnom cells, whether it is ssocited with HCC progression remins elusive. The results of the present study showed tht NG2 is useful s n dditionl prognostic mrker to increse the resolution of current pproches. Lu LL, Sun J, Li JJ, Jing Y, Bi LH, Zhng LD. Neuron-glil ntigen 2 overexpression in heptocellulr crcinom predicts poor prognosis. World J Gstroenterol 215; 21(21): Avilble from: URL: v21/i21/6649.htm DOI: i INTRODUCTION Heptocellulr crcinom (HCC) is common type of cncer, nd in Chin, its ssocited morbidity nd mortlity rtes hve become mong the highest in the world, ccounting nnully for 55% of new cses nd deths worldwide [1]. HCC tretment is extremely difficult becuse ptients re symptomtic until the cncer rpidly progresses into its terminl phse, creting chllenging nd ineffective tretment. In most cses, reltively fvorble curtive effects re chieved by surgicl liver resection, nd even fter liver resection, the 5-yer survivl rte is less thn 35% becuse of recurrence nd metstsis [2,3]. To help predict which ptients hve n unfvorble prognosis, it is criticl to identify dependble prognostic biomrker tht correltes with HCC progression, invsion, metstsis nd recurrence. Neuron-glil ntigen 2 (NG2)/chondroitin sulfte proteoglycn (CSPG-4) is n integrl membrne proteoglycn locted on the surfce of mny types of progenitor cells [4]. NG2 is phylogeneticlly conserved protein [5] composed of 28-kD N-linked glycoprotein nd 45-kD proteoglycn [6] tht plys numerous meditory roles in the prolifertion, migrtion, nd metstsis of melnom cells [7]. The humn homologue of NG2 is expressed in most humn melnoms nd promotes the growth nd metstsis of melnom cells [7]. NG2 expression is ssocited with metstsis in soft-tissue srcom ptients [8] nd with the progression of gliom [9], strocytom [1], nd myeloid leukemi [11]. NG2 lso regultes cell dhesion to collgen nd other extrcellulr mtrix (ECM) components [12-14]. NG2 is expressed on the surfce of differentited mlignnt cells, progenitor cells, nd cncer stem cells in vrious tumors [15]. Furthermore, NG2 plys criticl role in the growth, migrtion, nd metstsis of tumor cells [8]. In the norml dult brin, NG2 is expressed by 5% of ll neurl cells, hs wide ntomicl distribution, nd is expressed in more thn 7% of cycling progenitors. NG2+ progenitors hve the bility to proliferte, self-renew, nd produce different types of neurl cells under norml nd pthologicl conditions. Collectively, these chrcteristics hve led to reevlution of the linege potentil nd function of the previously considered unipotent oligodendrocyte precursors. It remins unknown whether HCC migrtion nd metstsis cn be ttributed to NG2 expression nd function. Thus, this study investigted whether NG2 is overexpressed in HCC, whether it is ssocited with HCC progression, invsion, metstsis, nd recurrence, nd whether it could be n effective prognostic mrker. 665 June 7, 215 Volume 21 Issue 21

3 Lu LL et l. NG2 expression in heptocellulr crcinom Tble 1 Reltionship between neuron-glil ntigen 2 expression nd clinicopthologicl chrcteristics of heptocellulr crcinom ptients Chrcteristic n NG2 χ 2 P vlue High Low Heptocellulr crcinom <.1 Non-tumor liver Totl cses Age (yr) < Sex Mle Femle Tumor size 5 cm < 5 cm TNM stge Ⅰ, Ⅱ Ⅲ, Ⅳ Tumor differentition Well Moderte Poor Serum AFP 2 ng/dl < 2 ng/dl Recurrence Yes No Invsion Yes No Tumor number Multiple Single Tumor cpsule Yes No AFP: Alph fetoprotein; NG2: Neuron-gli ntigen 2; HCC: Heptocellulr crcinom; TNM stge: Tumor node metstsis stge. MATERIALS AND METHODS Tissue smples All ptients provided written informed consent to use tumor tissue for clinicl reserch. Tissue smples were collected from 132 HCC ptients who underwent heptectomy t the Deprtment of Heptobiliry Surgery between Jnury nd December 27, ccording to protocols pproved by the Medicl Ethics Committee of the Southwest Hospitl nd the First Affilited Hospitl of the Third Militry Medicl University. No ptients received rdiotherpy or chemotherpy before heptectomy. The survivors were followed for 6 yers. The tumors were pthologiclly grded ccording to the World Helth Orgniztion clssifiction system, nd the pthologicl stging fter heptectomy ws performed ccording to the 22 tumor-nodemetstsis (TNM) clssifiction of mlignnt tumors from the Interntionl Union Aginst Cncer. The clinicopthologicl chrcteristics of the ptients re listed in Tble 1. Immeditely fter resection, 3 fresh tumor tissue specimens were snp-frozen in liquid nitrogen nd stored t -8. Overll survivl (OS) ws defined s the period between surgery nd deth or the lst contct. Survivor dt t the lst followup were bridged. Relpse-free survivl (RFS) ws defined s the period between surgery nd ny form of recurrence [16]. Immunohistochemistry Tissue sections were deprffinized in xylene nd rehydrted in ethnol. In vivo peroxidse ctivity ws blocked with 3% H2O2 for 1 min. The sections were subjected to ntigen retrievl in microwve oven in 1 mmol/l citrte buffer (ph 6.). The sections were incubted in 1% got serum lbumin in phosphtebuffered sline (PBS) for 3 min, then in n ntihumn NG2 polyclonl ntibody (1:1 dilution; Abcm, Burlingme, United Sttes) t 4 overnight, nd finlly with pproprite secondry ntibodies for 6 min nd wshed with PBS. Negtive controls were incubted with PBS insted of the primry ntibody under identicl conditions. RNA extrction nd quntittive rel-time polymerse chin rection (qrt-pcr) Totl RNA ws extrcted with the TRIzol regent (TKR, Dlin, Chin). qrt-pcr ws performed using the SYBR Green PCR Mster Mix (TKR, Dlin, Chin) on Rotor-Gene 6 RT-PCR System (Applied Biosystems) with the following cycling conditions: 95 for 3 s nd 4 cycles of 95 for 5 s nd 6 for 6 s. The primer pir sequences re provided below: NG2 forwrd 5 -CCTGTCCTCACCAATGTCCTCCT-3, NG2 reverse 5 -GCTGGCACTGTTGAGACTCTTGAC-3, β-ctin forwrd 5 -ATAGCACAGCCTGGATAGCAACGTAC-3 nd β-ctin reverse 5 -CACCTTCTACAATGAGCTGCGTGTG-3. The β-ctin gene ws used s the reference control. The reltive mrna expression levels were clculted bsed on the Ct vlues using the 2 -ΔΔCt method. Ech experiment ws repeted in triplicte. Western blot Western blot ws performed s previously described [17]. After electrophoresis, the proteins were trnsferred to polyvinylidene fluoride membrne. The membrne ws blocked with 5% nonft milk in Tris-buffered sline with Tween (TBST) nd incubted with n nti-humn NG2 polyclonl ntibody (1:1 dilution; Abcm, Burlingme, United Sttes) in TBST contining 5% bovine serum lbumin t 4 overnight. After wshing three times with TBST, the membrne ws incubted with horserdish peroxidse-conjugted secondry ntibody diluted in TBST t room temperture for 2 h. The protein signls were visulized using n electrochemiluminescence system. The gry vlues were clculted using Imge J2x. The NG2 protein levels were normlized to β-ctin protein levels June 7, 215 Volume 21 Issue 21

4 Lu LL et l. NG2 expression in heptocellulr crcinom Tble 2 Immunohistochemicl scoring system Sttisticl nlysis Significnt reltionships between NG2 expression nd the clinicopthologicl prmeters were evluted using the χ 2 test or Fisher s exct test. Survivl rtes were clculted using the Kpln-Meier method nd compred using the log-rnk test. The independent prognostic fctors for survivl were identified by multivrite nlysis using the Cox proportionl hzrd regression model. All the nlyses were performed using SPSS 17. for Windows. RESULTS Score Percent positive % 1%-1% 1 11%-5% 2 51%-8% 3 > 8% 4 Stining intensity No stining Wekly stined 1 Modertely stined 2 Strongly stined 3 The neuron-glil ntigen 2 (NG2) expression score ws clculted by multiplying the percent positive score by the stining intensity score; these scores rnged from -12. The tumors were divided into the following groups: negtive (-, score of ), low expression (1+, scores 1-4), moderte expression (2+, scores 5-8), nd strong expression (3+, scores 9-12). The NG2 immunohistochemistry results were used to divide the smples into two groups: low expression ( nd 1+) nd high expression (2+ nd 3+). NG2 protein expression in HCC smples NG2 expression ws semi-quntittively scored from the immunohistochemistry (IHC) dt bsed on the number of positive cells nd the stining intensity (Tble 2). Smples were considered NG2-positive if either the cell nucleus or cytoplsm stined positive. As shown in Tble 1, high NG2 expression ws observed in significntly more primry tumor smples (63.6%; 84/132) compred with the djcent noncncerous tissue smples (28.1%; 27/96) (p <.1). NG2 expression ws determined by IHC in 5 representtive pirs of heptic crcinom tissue nd djcent nontumorous tissue (Figure 1). To confirm the NG2 expression levels observed by IHC, we mesured NG2 expression in 3 rndomly selected tumor smples by qrt-pcr nd Western blot. The results show tht NG2 protein nd mrna expression mrkedly incresed in primry tumor smples compred with the djcent noncncerous tissue smples (Figure 2). Assocition between NG2 protein expression nd the clinicopthologicl fetures of HCC As presented in Tble 1, the correltions between NG2 protein expression nd the clinicopthologicl fetures of HCC ptients were nlyzed using the χ 2 test. High NG2 protein expression ws closely ssocited with tumor differentition (p =.89), recurrence (p =.163), TNM stge (p =.27), nd invsion (p =.193). However, no significnt reltionship ws observed between NG2 protein expression in HCC nd other prmeters, such s ge, sex, tumor size, serum AFP, tumor number, or tumor cpsule. NG2 expression in humn heptic crcinom tissues t TNM Ⅲ/Ⅳ stges ws significntly incresed compred with TNM Ⅰ/Ⅱ stge tissues (Figure 3A). Additionlly, NG2 expression in humn heptic crcinom tissues with poor degree of differentition ws significntly incresed compred with moderte nd high degrees of differentition (Figure 3B). Moreover, NG2 expression incresed in humn heptic crcinom tissues with portl vein invsion (Figure 3C), nd interestingly, NG2 ws highly expressed in vessels within humn heptic crcinom tissue (Figure 3D). Correltion between NG2 protein expression nd ptient survivl To ssess the prognostic vlue of NG2 for HCC, the ssocition between NG2 expression nd survivl ws nlyzed using the Kpln-Meier method with the logrnk test. Ptients were seprted into two groups, n NG2-high group (n = 84) nd n NG2-low group (n = 48). The log-rnk test indicted significnt difference in the overll survivl of HCC ptients with high NG2 expression compred with those with low NG2 expression (p <.1; Figure 4A). Moreover, NG2 expression in HCC tissue significntly correlted with disese-free survivl (p <.1; Figure 4B). Other prognostic fctors were investigted, nd univrite nlysis reveled tht invsion, serum AFP nd TNM stge dversely ffected the disese-free nd overll survivl (Tble 3). Moreover, serum AFP nd tumor number dversely ffected progression-free but not overll survivl (Tble 3). Combintion nlysis of the survivl dt reveled tht 25 (48.1%) of the 52 HCC ptients with TNM stge Ⅰ/Ⅱ disese were NG2-positive nd hd n unfvorble outcome (Figure 5A nd B). In contrst, 59 (73.7%) of the 8 HCC ptients with TNM stge Ⅲ/Ⅳ disese were NG2-positive but hd n unfvorble outcome (Figure 5A nd B). Similrly, of the 9 ptients with low serum AFP, 54 (6%) hd NG2- positive tumors nd n unfvorble outcome (Figure 5C nd D). Interestingly, 51 (72.8%) of the 7 HCC ptients with venous invsion were NG2-positive nd hd poor outcome (Figure 5E nd F). Conversely, of the 62 ptients without venous invsion, 33 (53.2%) with NG2-positive tumors did not hve fvorble outcome (Figure 5E nd F). These results indicte tht high NG2 expression correlted with shorter survivl time. Cox regression nlysis To further evlute the prognostic vlue of NG2 expression, Cox multivrite proportionl hzrds regression nlysis ws performed with ll the vribles to derive risk estimtes relted to disese June 7, 215 Volume 21 Issue 21

5 Lu LL et l. NG2 expression in heptocellulr crcinom ANT T P.1 P.2 P.3 P.4 P.5 Figure 1 Neuron-glil ntigen 2 protein expression in humn heptic crcinom nd mtched djcent non-tumorous tissues. Neuron-glil ntigen 2 expression ws exmined immunohistochemiclly in 5 representtive pirs of heptic crcinom tissue (T) nd djcent non-tumorous tissue (ANT) (mgnifiction 1) June 7, 215 Volume 21 Issue 21

6 Lu LL et l. NG2 expression in heptocellulr crcinom A Reltive protein expression B NG2 β-ctin NG ANT T ANT T ANT T ANT T ANT T ANT T ANT T ANT T T ANT β-ctin C Reltive mrna expression D Reltive mrna expression T ANT. T ANT Figure 2 Neuron-glil ntigen 2 protein nd mrna expression in humn heptic crcinom nd mtched djcent non-tumorous tissues. A: The reltive neuron-glil ntigen 2 (NG2) protein expression levels were mesured by Western blot. NG2 expression significntly incresed in heptic crcinom tissue (T) compred with mtched djcent non-tumorous tissue (ANT) (n = 3). The horizontl lines represent the men NG2 protein expression level, nd the P vlue is from the pired smples t-test; B: Representtive Western blot imges of NG2 protein expression in 8 sets of humn heptic crcinom (T) nd mtched ANT tissues. β-ctin expression ws used s loding control; C: The reltive NG2 mrna expression levels significntly incresed in heptic crcinom tissue (T) compred with mtched ANT (n = 3). The mrna expression levels were mesured by RT-PCR. The horizontl lines represent the men NG2 mrna expression levels, nd the P vlue is from the pired smples t-test; D: NG2 mrna expression levels were compred in 8 pirs of heptic crcinom (T) nd mtched ANT tissues. P <.5. free nd overll survivl nd to control for confounders. Multivrite nlysis (Tble 4) of the bove-mentioned significnt indictors of OS nd DFS showed tht NG2 expression (p =.2), serum AFP (p =.3), TNM stge (p =.1), nd portl vein invsion (p =.2) were independent prognostic indictors for OS in HCC ptients. Furthermore, NG2 expression (p =.3), serum AFP (p =.8), TNM stge (p =.1), tumor cpsule (p =.45), nd portl vein invsion (p =.2) were independent prognostic indictors for DFS in HCC ptients (Tble 4). NG2 expression in HCC ws identified s powerful prognostic indictor of disesefree nd overll survivl. DISCUSSION This study is the first report to estblish NG2 s n independent nd powerful prognostic mrker for disese progression nd overll survivl for ptients with surgiclly resected primry HCC. In this study, we evluted NG2 expression t both the trnscriptionl nd trnsltionl levels in numerous heptocellulr crcinom tissue smples nd corresponding mtched djcent non-tumor tissue smples using qrt-pcr nd Western blot. We found tht NG2 mrna nd protein were significntly overexpressed in tumor tissue compred with mtched djcent non-tumorous tissue, which ws consistent with the IHC nlysis. These results suggested tht NG2 might ply n importnt role in HCC tumorigenesis. Indeed, NG2 expression hs been correlted with the progression of melnom [7], gliom [9], strocytom [1], nd myeloid leukemi [18]. We lso found tht high NG2 expression ws significntly correlted with poor tumor differentition, the presence of vsculr invsion, TNM stge nd recurrence. In HCC, the correltions between NG2 expression nd poor tumor differentition, TNM stge nd the presence of vsculr invsion re intriguing. Kpln-Meier survivl nlysis reveled significnt correltion between NG2 expression nd overll nd relpse-free survivl. A more comprehensive nlysis of the survivl dt reveled tht mong ptients with TNM stge Ⅲ/Ⅳ disese or the presence of vsculr invsion, most ptients lso hd NG2-positive tumors nd n unfvorble outcome. However, lrge proportion of the ptients with TNM stge Ⅰ/Ⅱ disese without vsculr invsion but with NG2-positive tumors still hd n unfvorble outcome. These results indicted tht the combintion of NG2 expression with current clinicopthologicl prognostic mrkers my ccurtely predict HCC ptient prognosis fter heptic resection. The Cox regression nlysis demonstrted 6654 June 7, 215 Volume 21 Issue 21

7 Lu LL et l. NG2 expression in heptocellulr crcinom A b c d B b c d C b c d D b c d Figure 3 Correltion between neuron-glil ntigen 2 expression nd clinicopthologicl fetures. A: Neuron-glil ntigen 2 (NG2) expression in humn heptic crcinom tissues t different clinicl stges (: TNM stge Ⅰ; b: TNM stgeⅡ; c: TNM stge Ⅲ; d: TNM stge Ⅳ); B: NG2 protein expression in humn heptic crcinom tissues with different degrees of differentition (: Normlly differentited; b: Well differentited; c: Modertely differentited; d: Poorly differentited); C: NG2 expression in humn heptic crcinom tissues with portl vein invsion [, c: Vsculr invsion (mgnifiction 1); b, d: Vsculr invsion (mgnifiction 4)]; D: NG2 expression in vessels within humn heptic crcinom tissue [, c: Tumor vessel (mgnifiction 2); b, d: Tumor vessel (mgnifiction 4)]. A 1 P <.1 B 1 P <.1 Overll survivl (%) Low NG2 expression (n = 48) Recurrence-free survivl (%) Low NG2 expression (n = 46) High NG2 expression (n = 84) High NG2 expression (n = 75) Figure 4 Kpln-Meier curves of overll survivl (A) nd disese-free survivl (B) bsed on neuron-glil ntigen 2 expression in heptocellulr crcinom ptients. The P-vlues were determined using the log-rnk test June 7, 215 Volume 21 Issue 21

8 Lu LL et l. NG2 expression in heptocellulr crcinom A 1 B 1 Overll survivl (%) b c d Disese-free survivl (%) d b c C 1 D 1 Overll survivl (%) b c d Disese-free survivl (%) d b c E 1 F 1 Overll survivl (%) d b c Disese-free survivl (%) d b c Figure 5 combintion nlysis of the survivl dt. Overll survivl (A) nd disese-free survivl (B) in heptocellulr crcinom ptients bsed on NG2 expression nd TNM stge. : TNM stges Ⅰ/Ⅱwith NG2 low expression; b: TNM stges Ⅰ/Ⅱwith NG2 high expression; c: TNM stges Ⅲ/Ⅳ with NG2 low expression; d: TNM stges Ⅲ/Ⅳ with NG2 high expression; Overll survivl (C) nd disese-free survivl (D) in heptocellulr crcinom ptients bsed on NG2 expression nd serum AFP. : Low level of serum AFP with NG2 low expression; b: Low level of serum AFP with NG2 high expression; c: High level of serum AFP with NG2 low expression; d: High level of serum AFP with NG2 high expression; Overll survivl (E) nd disese-free survivl (F) in heptocellulr crcinom ptients bsed on NG2 expression nd venous invsion. : No invsion with NG2 low expression; b: No invsion with NG2 high expression; c: Invsion with NG2 low expression; d: No invsion with NG2 high expression. tht NG2 expression ws significnt independent predictor of OS nd DFS in HCC ptients fter heptectomy, incresing its promise s predictive biomrker. NG2 might ply role in promoting HCC progression nd metstsis. Mny different signling mechnisms hve been proposed by which NG2 could contribute to mlignnt trnsformtion nd subsequently promote 6656 June 7, 215 Volume 21 Issue 21

9 Lu LL et l. NG2 expression in heptocellulr crcinom Tble 3 Univrite nlysis of prognostic fctors for disesefree survivl nd overll survivl in 132 ptients with heptocellulr crcinom Fctor n 6-yr disese-free survivl P vlue 6-yr overll survivl P vlue NG2.. Negtive % 29.2% Positive % 9.5% Age (yr) % 21.% < % 31.4% Sex Mle % 23.6% Femle % 38.5% Tumor size cm % 28.9% < 5 cm % 21.4% TNM stge.1.3 Ⅰ, Ⅱ % 34.6% Ⅲ, Ⅳ 8 9.7% 21.3% Differentition Well % 33.3% Moderte 52 2.% 9.6% Poor % 12.8% Serum AFP ng/dl % 18.% < 2 ng/dl % 14.% Invsion.. Yes 7 7.7% 12.9% No % 21.% Number Multiple % 16.1% Single % 16.8% Cpsule Yes % 21.6% No % 32.8% NG2: Neuron-gli ntigen 2; TNM stge: Tumor node metstsis stge. tumor formtion nd metstsis. The complex mechnisms by which NG2 ffects melnom progression hve strted to be defined, in prticulr the ssocition with other cell surfce proteins nd receptor tyrosine kinses (RTKs) nd its centrl role in modulting the function of these proteins [19]. NG2 is essentil to the growth of melnom tumors through its modultion of integrin function nd enhnced growth fctor receptor-regulted pthwys including sustined ctivtion of ERK 1/2. The ctivtion of integrin, RTK, nd ERK 1/2 function by NG2 modultes numerous spects of tumor progression [2,21]. Expression of fulllength NG2 in RGP humn melnom cells lcking endogenous NG2 expression results in significntly enhnced integrin medited spreding nd FAK ctivtion [22]. As NG2 cn ctivte MMP complexes on melnom cell surfce, the collective results implicte NG2 s n importnt contributing fctor to loclized invsion t the leding edge of invsive primry tumors [23-25]. Finlly, NG2-medited ctivtion of integrin fcilittes enhnced survivl of humn glioblstom cells when they were exposed to cliniclly used chemotherpeutic gents by enhncing sustined ctivtion of AKT vi PI3K [22]. Tble 4 Multivrite nlysis of individul prmeters correlting with overll survivl nd disese-free survivl Vrible Overll survivl Disese-free survivl NG2 overexpression my induce highly ggressive tumors chrcterized by incresed ngiogenesis nd modertely invsive phenotype [26,27]. Indeed, NG2- positive tumors grow more rpidly, hve disrupted blood-brin brrier integrity, nd exhibit n incresed vsculr volume frction [26,28]. The NG2 proteoglycn, whose extended centrl D2 domin binds to type Ⅵ collgen, cts s link between the cell surfce nd the extrcellulr mtrix [13]. Similr results hve been chieved on lminin 2-coted surfces [29,3]. The roles of collgen Ⅵ nd lminin 2 in the brin vsculture nd their ssocition with xonl processes enble the migrtion of NG2-positive gliom cells long blood vessels nd nerve fiber trcts [31,32]. This study is limited by its retrospective nture nd the inclusion of only ptients with resectble tumors. It is possible tht these findings pertin only to those ptients who hve undergone resection. Lrger prospective studies exmining NG2 re necessry to further vlidte the usefulness of this system. Suppressing NG2 overexpression my prevent invsive progression nd metsttic relpse, which would improve the prognosis nd qulity of life for HCC ptients fter heptic resection. In the ner future, trgeting the NG2 pthwy my be therpeutic strtegy for HCC tht ssists conventionl chemotherpy or rdiotherpy. NG2 might ply role in promoting HCC progression nd metstsis. However, current studies on the functionl roles of NG2 re superficil, nd future studies should concentrte on the moleculr mechnisms by which NG2 contributes to HCC development nd progression. COMMENTS HR (95%CI) P vlue HR (95%CI) P vlue TNM 2.39 ( ) ( ).1 stge Serum 1.93 ( ) ( ).8 AFP Tumor ( ).45 cpsule Invsion ( ) ( ).2 NG ( ) ( ).3 TNM stge: Tumor node metstsis stge; AFP: Alph fetoprotein; NG2: Neuron-gli ntigen 2. Bckground Neuron-glil ntigen 2 (NG2)/chondroitin sulfte proteoglycn is n integrl membrne proteoglycn locted on the surfce of mny types of progenitor cells. NG2 is phylogeneticlly conserved protein composed of 28-kD N-linked glycoprotein nd 45-kD proteoglycn tht plys numerous meditory roles in the prolifertion, migrtion, nd metstsis of melnom cells. NG2 is expressed on the surfce of differentited mlignnt cells, progenitor cells, nd cncer stem cells in vrious tumors. Furthermore, NG2 plys criticl role in the growth, migrtion, nd metstsis of tumor cells June 7, 215 Volume 21 Issue 21

10 Lu LL et l. NG2 expression in heptocellulr crcinom Whether NG2 is overexpressed in heptocellulr crcinom (HCC), whether it is ssocited with HCC progression, invsion, metstsis, nd recurrence, nd whether it could be n effective prognostic mrker remin unknown. Reserch frontiers This study utilized immunohistochemistry (IHC) to guge NG2 expression semiquntittively bsed on the number of positive cells nd the stining intensity. To confirm the NG2 expression levels observed by IHC, we mesured NG2 expression in 3 rndomly selected tumor nd djcent noncncerous tissue smples by qrt-pcr nd Western blot. Those techniques re highly specific nd efficient, esy to control nd mnipulte, verstile, nd time sving. Innovtions nd brekthroughs This study demonstrtes tht NG2 expression in primry tumor smples ws significntly more thn tht in the djcent noncncerous tissue smples. In ddition, it demonstrted significnt difference in the overll survivl of HCC ptients with high NG2 expression compred with those with low NG2 expression. Furthermore, NG2 expression ws n independent prognostic indictor for DFS in HCC ptients. Applictions The results of the present study suggest tht NG2 expression in HCC ws identified s powerful prognostic indictor of disese-free nd overll survivl. Terminology Overll survivl ws defined s the period between surgery nd deth or the lst contct. Survivor dt t the lst follow-up were bridged. Relpse-free survivl ws defined s the period between surgery nd ny form of recurrence. Peer-review The study NG2 overexpression in HCC predicts poor prognosis is n ttrctive nd interesting study, lthough, s noted by the uthor, it remins retrospective nd is focused only on subjects who underwent surgicl resection. REFERENCES 1 Yng ZQ, Yng ZY, Zhng LD, Ping-Bie SG, M KS, Li XW, Dong JH. 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Angiogenesis 214; 17: [PMID: DOI: 1.17/s ] 22 Chekeny M, Krkstd C, Svendsen A, Netlnd IA, Stlesen V, Tysnes BB, Selheim F, Wng J, Skrissen PØ, Sndl T, Lønning PE, Fltmrk T, Enger PØ, Bjerkvig R, Sioud M, Stllcup WB. The progenitor cell mrker NG2/MPG promotes chemoresistnce by ctivtion of integrin-dependent PI3K/Akt signling. Oncogene 28; 27: [PMID: DOI: 1.138/ onc ] 23 Liu H, Shubyev VI. Mtrix metlloproteinse-9 controls prolifertion of NG2+ progenitor cells immeditely fter spinl 6658 June 7, 215 Volume 21 Issue 21

11 Lu LL et l. NG2 expression in heptocellulr crcinom cord injury. Exp Neurol 211; 231: [PMID: DOI: 1.116/j.expneurol ] 24 Joo NE, Mio D, Bermúdez M, Stllcup WB, Kpil YL. Shedding of NG2 by MMP-13 ttenutes noikis. DNA Cell Biol 214; 33: [PMID: DOI: 1.189/dn ] 25 Schultz N, Nielsen HM, Minthon L, Wennström M. Involvement of mtrix metlloproteinse-9 in myloid-β 1-42-induced shedding of the pericyte proteoglycn NG2. J Neuropthol Exp Neurol 214; 73: [PMID: DOI: 1.197/ NEN.84] 26 Wng J, Svendsen A, Kmiecik J, Immervoll H, Skftnesmo KO, Plngumà J, Reed RK, Bjerkvig R, Miletic H, Enger PØ, Rygh CB, Chekeny M. Trgeting the NG2/CSPG4 proteoglycn retrds tumour growth nd ngiogenesis in preclinicl models of GBM nd melnom. PLoS One 211; 6: e2362 [PMID: DOI: /journl.pone.2362] 27 Vn Sinderen M, Cumn C, Winship A, Menkhorst E, Dimitridis E. The chrondroitin sulfte proteoglycn (CSPG4) regultes humn trophoblst function. Plcent 213; 34: [PMID: DOI: 1.116/j.plcent ] 28 Girolmo F, Dlltomsin A, Rizzi M, Errede M, Wälchli T, Mucignt MT, Frei K, Roncli L, Perris R, Virgintino D. Diversified expression of NG2/CSPG4 isoforms in glioblstom nd humn foetl brin identifies pericyte subsets. PLoS One 213; 8: e84883 [PMID: DOI: /journl.pone.84883] 29 Done KJ, Howell SJ, Birk DE. Identifiction nd functionl chrcteriztion of two type VI collgen receptors, lph 3 bet 1 integrin nd NG2, during vin cornel stroml development. Invest Ophthlmol Vis Sci 1998; 39: [PMID: ] 3 Iid J, Meijne AM, Spiro RC, Roos E, Furcht LT, McCrthy JB. Spreding nd focl contct formtion of humn melnom cells in response to the stimultion of both melnom-ssocited proteoglycn (NG2) nd lph 4 bet 1 integrin. Cncer Res 1995; 55: [PMID: ] 31 Ay-y J, Myer J, Ekin AK, Muffly BG, Anello M, Sndy JD, Gottschll PE. The effect of hypoxic-ischemic brin injury in perintl rts on the bundnce nd proteolysis of brevicn nd NG2. Exp Neurol 25; 193: [PMID: DOI: 1.116/j.expneurol ] 32 Morit S, Houri A, Miyt S. Chnges in pericytic expression of NG2 nd PDGFRB nd vsculr permebility in the sensory circumventriculr orgns of dult mouse by osmotic stimultion. Cell Biochem Funct 214; 32: [PMID: DOI: 1.12/cbf.2971] P- Reviewer: Berkne S, Tomizw M, Zhng ZM S- Editor: M YJ L- Editor: Wng TQ E- Editor: Liu XM 6659 June 7, 215 Volume 21 Issue 21

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