分子诊断在胶质瘤分类中的进展
2017-01-12魏明海胡增春
马 辉,魏明海,胡增春
(大连医科大学附属第二医院:1药剂科,2神经外科,辽宁 大连116033)
分子诊断在胶质瘤分类中的进展
马 辉1,魏明海2,胡增春2
(大连医科大学附属第二医院:1药剂科,2神经外科,辽宁 大连116033)
胶质瘤的治疗及预后面临巨大挑战,最近随着关键基因和分子标记的发现,WHO更新了胶质瘤的诊断依据和分类标准.这些更新(IDH、1p/19q联合性缺失、K27M变异等)要求系统回顾既往病例资料和对相关临床治疗和基础研究进行重新思考和设计.本文总结了最新胶质瘤分类的决定基因和分子标志,以及相应的临床治疗方案选择.
胶质瘤;分子诊断;分类;治疗;预后
0 引言
2007版WHO分类中神经胶质肿瘤包括星形细胞瘤、少突胶质细胞瘤、少突星形细胞瘤、室管膜瘤、神经元和神经元⁃胶质混合肿瘤[1],这个分类包括了从Ⅰ级(如毛细胞型星形细胞瘤)至Ⅳ级(如胶质瘤母细胞瘤/glioblastoma,GBM)的各级别胶质瘤.而这些“金标准”仍存在缺陷,例如这些诊断是依赖于观察者的经验给出的,不同病理学家间可能存在鉴别诊断差异.另外,组织学诊断与预后相关性较差,同一种病理类型的患者即使排除术前一般情况、肿瘤部位、大小及切除程度等因素后,其预后仍可能存在几个月至几年的差异.随着对胶质瘤生物学行为的深入研究,尤其是关键基因和分子的发现,2016年 WHO在原有组织学分类基础上,加入了分子诊断标志物[2].其中一个重要的更新是对侵袭性胶质瘤加入了分子诊断标准,这些分子诊断标准(如IDH突变、染色体1p/19q共缺失、组蛋白突变等)补充了2007版中组织学亚型的分类(如星形胶质细胞、少突胶质细胞瘤、少突星形胶质细胞等)[3].如新版引入弥漫性胶质瘤(diffuse glioma)概念代替2007版中依据细胞来源分类的各亚型,包括WHOⅡ、Ⅲ级星形细胞瘤、少突胶质细胞瘤、GBM、中线胶质瘤和儿童弥漫性胶质瘤.胶质瘤脑病不再作为一个独立的亚型,而被认为仅是许多神经胶质肿瘤的一种生长方式.针对星形细胞瘤,新版删除了原浆型和纤维型星形细胞瘤2个亚型(大部分为普通弥漫性星形细胞瘤),而将肥胖型星形细胞瘤归入IDH突变型.IDH突变的发生率在星形细胞瘤和GBM中有差别,IDH野生型多见于原发性GBM.少突胶质瘤细胞被定义为IDH突变型和1p/19q共缺失型,弥漫性中线胶质瘤被定义为组蛋白H3K27M突变型.如果分子检测结果不能获得,则按2007版组织学分类,但需表明NOS(not otherwise specified)[4].这些更新的依据是新版各亚组中肿瘤生长方式、临床生物学行为、基因突变和预后相似.本文旨在介绍新版分类中重要的基因和分子,探讨其在胶质瘤诊断、治疗及预后方面的作用.
1 IDH 突变(异柠檬酸脱氢酶/isocitrate dehydrogenase,IDH)
IDH突变后催化α⁃酮戊二酸转变为2⁃羟戊二酸(2HG)同时消耗NAD(P)H产生NAD(P),2HG被推测为一种癌代谢产物[5].IDH基因突变多出现在IDH1 132位点由精氨酸变为组氨酸(R132H)或IDH2 172位点由精氨酸变为赖氨酸(R172K).IDH突变被认为是相对早期事件[6],在星形细胞瘤中继发TP53和ATRX突变,在少突胶质细胞瘤中继发1p/19q共缺失[5].90%胶质瘤中发现IDH1 R132H突变[6-7].65%~80%Ⅱ~Ⅲ胶质瘤、80%~90%继发GBM存在IDH突变,而在原发GBM和儿童患者中则罕见[8-10].IDH突变型的预后优于 IDH 野生型[11],IDH野生型低级别胶质瘤侵袭性和预后甚至类似于GBM[12].IDH突变检测已成为胶质瘤分类的重要指标,提示着患者的预后.最近,针对IDH突变靶点的抗肿瘤免疫治疗显示延长了小鼠生存期[13],通过小分子IDH抑制物或IDH靶点疫苗研究已进入临床试验[14],未来针对IDH突变的各种治疗可能成为新的治疗靶点.
2 TP 53
超过50%的弥漫性星形细胞瘤、间变星形细胞瘤和继发GBM中发现TP53基因突变,但较少出现在少突胶质细胞瘤中[15].大多数侵袭性星形细胞瘤同时表达IDH变异和TP53变异,反之IDH野生型也较少表达TP53变异[9,16].因此在弥漫性星形细胞瘤中IDH变异和TP53变异有密切的联系.其强染色倾向于星形胶质细胞诊断.其免疫染色针对的是正常的P53蛋白,由于TP53变异导致P53蛋白寡聚体降解减少,故而其过表达常提示存在基因变异[17].但它并不是特异性指标,因为P53蛋白也可由其它机制上调.
3 染色体1p/19q共缺失型
染色体1p和19q臂缺失是由于易位不平衡导致形成der(1;19)(q10,p10)[18],继发染色体1p和19q上各自的肿瘤抑制基因远上游结合蛋白1(far⁃up⁃stream binding protein 1,FUBP1)和果蝇同系物(hom⁃olog of Drosophila capicua,CIC)失活[19].1p/19q共缺失型少突胶质瘤中30%存在FUBP1突变,83%存在CIC突变[20].在新版胶质瘤分类中,染色体1p/19q共缺失合并IDH突变被定义为少突胶质细胞瘤的一个亚型[2].染色体1p/19q共缺失型间变胶质瘤患者对放疗显示了良好的预后[21].染色体1p/19q共缺失型间变少突胶质细胞瘤单独放疗生存期明显长于完整型少突胶质细胞瘤(7.3年∶2.7年),使用PCV(甲苄肼、洛莫司汀、新长春碱)化疗方案可加倍延期前者生存期至14.7年[22].多个临床研究显示1p/19q共缺失型间变胶质细胞瘤患者PCV或替莫唑胺化疗均显示了良好的效果[21,23].1p/19q共缺失对于治疗和预后均是一个非常有用的指标,对于1p/19q共缺失型间变胶质瘤患者应常规使用放化疗.
4 ATRX和TERT
维持端粒是癌细胞避免衰老和保持增殖能力所必须的,胶质瘤细胞通过ATRX(α地中海贫血/精神延迟伴 X染色体综合征蛋白,α thalassemia/mental retardation syndrome Xlinked protein)维持端粒延长表型[lengthening of telomeres(ALT)phenotype]或通过TERT(端粒酶逆转录酶,Telomerase reverse tran⁃ scriptase)增加端粒表达,以便促进细胞存活或增殖能力.ATRX是一个在染色体重塑中非常重要的解螺旋酶.ATRX基因失活突变与ALT表型关系密切[24],仅出现在没有1p/19q共缺失型胶质瘤中[25].其突变合并IDH和TP53突变可诊断为星形细胞瘤(Ⅱ、Ⅲ星形细胞瘤或继发GBM),而野生型ATRX合并IDH突变和1p/19q共缺失则可诊断为少突胶质细胞瘤[25].前期研究显示抑制ATRX能够导致ALT瓦解,最终使染色体破裂细胞死亡[26].TERT是端粒酶的一个亚基,能够添加核苷酸至端粒.TERT基因在成人是失活状态,但在癌细胞中被重新激活,从而促进细胞存活和增殖.胶质瘤中TERT突变出现在TERT启动子228和250号位点.TERT突变主要发生在少突胶质细胞瘤和原发性GBM,常合并1p/19q共缺失,而星形胶质细胞和继发GBM则少见[27],这与ATRX相反.TERT突变对于诊断和预后非常有价值.超过1000例样本的资料分析显示,根据IDH突变、1p/19q共缺失和TERT突变这三个分子标记物可将胶质瘤患者分为5组:①3个分子均突变(三阳性)的LGGs为少突胶质细胞瘤,预后最好;②TERT和IDH突变,合并1p/19q完整型的LGGs(Ⅱ、Ⅲ级)预后类似三阳性组,Ⅳ级侵袭性明显,预后差;③仅IDH突变者为星形胶质细胞,预后居中,仍可存活数年;④仅TERT突变者为GBM(即使组织学为LGGs),预后最差;⑤野生型TERT和IDH,合并1p/19q完整型(三阴性)为青年GBM,预后比①和②组差,但优于④组[25].最近有研究显示TERT突变合并MGMT甲基化能够增加替莫唑胺敏感性,延长生存期,但其突变若合并MGMT非甲基化则增加了化疗耐药性,预后更差[28],这种分歧化的效应及与MGMT甲基化之间的关系仍有待进一步研究.
5 组蛋白突变
有研究显示儿童患者胶质瘤中存在由HIST1H3B和H3F3A基因编码的组蛋白3.1和组蛋白3.3变异.这些蛋白由异染色体DNA结构变化调节,指导转录激动子和受体的相互作用,在转录后的表观遗传学表达中发挥重要作用,同时也调节端粒.78%弥漫性内生型桥脑胶质瘤(diffuse intrinsic pon⁃tine gliomas,DIPGs)和22%脑干外生型GBM中存在由H3F3A基因突变产生的K27M变异[29].H3F3A基因突变多见于儿童及青年胶质瘤患者(5~23岁),多发生于中线部位,如丘脑、脑桥、脊髓,特别是DIPGs.H3F3A基因突变通常合并TP53、ATRX和DAXX等其他关键基因突变.含有H3F3A突变所致的K27变异的DIPGs对比野生型DIPGs常显示更差的预后[30].未来,针对这些突变靶点可能研制新的抗原受体T细胞治疗胶质瘤[31].
6 MGMT
MGMT(6⁃甲基鸟嘌呤甲基转移酶,O6⁃methylgua⁃nine methyltransferase,MGMT)是一种从鸟嘌呤上切除烷基的DNA错配修复酶[32].这一过程涉及如替莫唑胺和洛莫司汀等烷基化疗药物的耐药性.MGMT的高甲基化能表观遗传学沉默MGMT蛋白,使其表达减少,从而提高烷基药物的反应性.因此,当使用烷基药物治疗含有MGMT启动子CpG位点甲基化的胶质瘤对比非甲基化患者可以产生更长的无进展期和生存期(21.7月 ∶12.7月)[33].约40%GBM含有MGMT启动子甲基化[34].现在很多中心对GBM常规检测MGMT启动子甲基化.多项研究表明,针对MGMT非甲基化的老年胶质瘤患者应用替莫唑胺等烷基化疗药物并没有治疗获益[35-36].虽然MGMT启动子甲基化对药物选择和预后很有帮助,但由于其在各个基因亚型中变异较大,故在新版WHO分类中没有将其作为诊断亚型的依据.MGMT启动子甲基化作为表观遗传学的一个有价值的治疗靶点在未来应该给予更多关注.
7 BRAF
RAF家族在哺乳动物中有 A⁃RAF、B⁃RAF、C⁃RAF 3个异构体.BRAF是丝氨酸/苏氨酸蛋白激酶,通过Ras/Raf/MEK/ERK途径调节细胞外激酶,在细胞分裂、生长和增殖中扮演重要角色[37].60%毛细胞型星形细胞瘤含有 BRAF基因和 KIAA1549基因的融合,导致BRAF激活[38].常见融合位点在KIAA1549基因外显子16和BRAF外显子9(KEx16BEx9).而BRAF V600E突变则是在600位点上谷氨酸替代缬氨酸,导致MEK/ERK激活,刺激细胞增殖和存活.这种突变主要发生于多形性黄色星形细胞瘤(60%)和神经节细胞胶质瘤(50%),常提示与肿瘤进展和复发高度相关[39].针对含有BRAF V600E突变的胶质瘤患者,对其使用靶向药物可能是有效的.其抑制剂拉菲尼和维罗非尼已在黑色素瘤治疗上取得成功[40],目前相关研究多为个案研究[41-42].已有维罗非尼用于治疗含有BRAF V600E突变的儿童上皮型GBM获得长期无进展生存期报道[43],进一步的临床试验研究已经开展[44].故BRAF V600E可能是某些胶质瘤亚型治疗的一个有希望的靶点.
8 侵袭性胶质瘤全部分子资料收集
癌症基因组地图网络(The cancer genome atlas network,TCGA)回顾整理了美国多中心肿瘤样本,使用传代测序、微阵列芯片检测基因表达、RNA测序、全基因甲基化阵列、反转溶解产物蛋白分析等技术检测了低级别胶质瘤(low grade gliomas,LGGs)和GBM的全部分子特征[45].这些研究发现LGGs可分成3个分子亚组:①IDH1/IDH2突变、DNA高度甲基化合并染色体1p/19q共缺失型,对应少突胶质细胞瘤,这类肿瘤有较好的预后,中位生存期达7年;②IDH1/IDH2突变、DNA高度甲基化合并染色体1p/19q完整型,常合并ATRX和TP53改变,对应星形细胞瘤,中位生存期约5年;③IDH1/IDH2野生型合并染色体1p/19q完整型,即使一些组织学显示低级别,但其行为学类似“前胶质母细胞瘤”状态,中位生存期仅约1.7年[46].这些研究促成WHO最近对胶质瘤和其他一些肿瘤分类的修订.
依赖于基因组学、表观遗传学和蛋白质组学的胶质瘤亚型分类已进入了一个新时期,随着相关研究的深入,不仅有助于诊断分类,更有助于进一步深刻地理解各类型胶质瘤的生物学行为,研发新的抗肿瘤药物,指导选择治疗方案并对判断预后提供帮助,以便最终为胶质瘤患者提供最佳的个体化治疗方案.
[1]Louis DN,Ohgaki H,Wiestler OD,et al.The 2007 WHO classifica⁃tion of tumours of the central nervous system[J].Acta Neuropathol,2007,114(2):97-109.
[2]Louis DN,Perry A,Reifenberger G,et al.The 2016 World Health Organization classification of tumors of the central nervous system:a summary[J].Acta Neuropathol,2016,131(6):803-820.
[3]Bush NA,Butowski N.The effect of molecular diagnostics on the treatment of glioma[J].Curr Oncol Rep,2017,19(4):26.
[4]Chen R,Smith⁃Cohn M,Cohen AL,et al.Glioma Subclassifications and Their Clinical Significance[J].Neurotherapeutics,2017,14(2):284-297.
[5]Thompson CB.Metabolic enzymes as oncogenes or tumor suppressors[J].N Engl J Med,2009,360(8):813-815.
[6]Suzuki H,Aoki K,Chiba K,et al.Mutational landscape and clonal architecture in gradeⅡandⅢ gliomas[J].Nat Genet,2015,47(5):458-468.
[7]Wakimoto H,Tanaka S,Curry WT,et al.Targetable signaling path⁃way mutations are associated with malignant phenotype in IDH⁃mu⁃tant gliomas[J].Clin Cancer Res,2014,20(11):2898-2909.
[8]Yang H,Ye D,Guan KL,et al.IDH1 and IDH2 mutations in tumorigenesis:mechanistic insights and clinical perspectives[J].Clin Cancer Res,2012,18(20):5562-5571.
[9]Yan H,Parsons DW,Jin G,et al.IDH1 and IDH2 mutations in gliomas[J].N Engl J Med,2009,360(8):765-773.
[10]Hartmann C,Meyer J,Balss J,et al.Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age:a study of 1,010 diffuse gliomas[J].Acta Neuropathol,2009,118(4):469-474.
[11]Wick W,Hartmann C,Engel C,et al.NOA⁃04 randomized phaseIII trial of sequential radiochemotherapy of anaplastic glioma with procarbazine,lomustine,and vincristine or temozolomide[J].J Clin Oncol,2009,27(35):5874-5880.
[12]Dunn GP,Andronesi OC,Cahill DP.From genomics to the clinic:biological and translational insights of mutant IDH1/2 in glioma[J].Neurosurg Focus,2013,34(2):E2.
[13]Pellegatta S,Valletta L,Corbetta C,et al.Effective immuno⁃targe⁃ting of the IDH1 mutation R132H in a murine model of intracranial glioma[J].Acta Neuropathol Commun,2015,3:4.
[14]Schumacher T,Bunse L,Pusch S,et al.A vaccine targeting mutant IDH1 induces antitumour immunity[J].Nature,2014,512(7514):324-327.
[15]Okamoto Y,Di Patre PL,Burkhard C,et al.Population⁃based study on incidence,survival rates,and genetic alterations of low⁃grade diffuse astrocytomas and oligodendrogliomas[J].Acta Neuropathol,2004,108(1):49-56.
[16]Watanabe T,Nobusawa S,Kleihues P,et al.IDH1 mutations are early events in the development of astrocytomas and oligodendroglio⁃mas[J].Am J Pathol,2009,174(4):1149-1153.
[17]Appin CL,Brat DJ.Molecular genetics of gliomas[J].Cancer J,2014,20(1):66-72.
[18]Jenkins RB,Blair H,Ballman KV,et al.A t(1;19)(q10;p10)mediates the combined deletions of 1p and 19q and predicts a better prognosis of patients with oligodendroglioma[J].Cancer Res,2006,66(20):9852-9861.
[19]Bettegowda C,Agrawal N,Jiao Y,et al.Mutations in CIC and FUBP1 contribute to human oligodendroglioma[J].Science,2011,333(6048):1453-1455.
[20]Yip S,Butterfield YS,Morozova O,et al.Concurrent CIC mutations,IDH mutations,and 1p/19q loss distinguish oligodendrogliomas from other cancers[J].J Pathol,2012,226(1):7-16.
[21]Kaloshi G,Benouaich⁃Amiel A,Diakite F,et al.Temozolomide for low⁃grade gliomas:Predictive impact of 1p/19q loss on response and outcome[J].Neurology,2007,68(21):1831-1836.
[22]Cairncross G,Wang M,Shaw E,et al.Phase III trial of chemoradio⁃therapy for anaplastic oligodendroglioma:long⁃term results of RTOG 9402[J].J Clin Oncol,2013,31(3):337-343.
[23]van den Bent MJ,Brandes AA,Taphoorn MJB,et al.Adjuvant procar⁃bazine,lomustine,and vincristine chemotherapy in newly diagnosed anaplastic oligodendroglioma:long⁃term follow⁃up of EORTC Brain Tumor Group study 26951[J].J Clin Oncol,2013,31(3):344-350.
[24]Heaphy CM,de Wilde RF,Jiao Y,et al.Altered telomeres in tumors with ATRX and DAXX mutations[J].Science,2011,333(6041):425.
[25]Eckel⁃Passow JE,Lachance DH,Molinaro AM,et al.Glioma groups based on 1p/19q,IDH,and TERTpromoter mutations in tumors[J].N Engl J Med,2015,372(26):2499-2508.
[26]Flynn RL,Cox KE,Jeitany M,et al.Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors[J].Science,2015,347(6219):273-277.
[27]Koelsche C,Sahm F,Capper D,et al.Distribution of TERT promot⁃er mutations in pediatric and adult tumors of the nervous system[J].Acta Neuropathol,2013,126(6):907-915.
[28]Arita H,Yamasaki K,Matsushita Y,et al.A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas[J].Acta Neuro⁃pathol Commun,2016,4(1):79.
[29]Wu G,Broniscer A,McEachron TA,et al.Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non⁃brainstem glioblastomas[J].Nat Genet,2012,44:251-253.
[30]Khuong⁃Quang DA,Buczkowicz P,Rakopoulos P,et al.K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas[J].Acta Neuropathol,2012,124(3):439-447.
[31]Kohanbash G,Okada K,Liu S,et al.HG⁃81 novel and shared neoantigen for glioma T cell therapy derived from histone 3 variant H3.3 K27M mutation[J].Neuro Oncol,2016,18:iii67.
[32]Hegi ME,Diserens AC,Gorlia T,et al.MGMT gene silencing and benefit from temozolomide in glioblastoma[J].N Engl J Med,2005,352(10):997-1003.
[33]Stupp R,Hegi ME,Mason WP,et al.Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study:5⁃year analysis of the EORTC⁃NCIC trial[J].Lancet Oncol,2009,10(5):459-466.
[34]Esteller M,Garcia⁃Foncillas J,Andion E,et al.Inactivation of the DNA⁃repair gene MGMT and the clinical response of gliomas to alky⁃lating agents[J].N Engl J Med,2000,343:1350-1354.
[35]Malmström A,Grønberg BH,Marosi C,et al.Temozolomide versus standard 6⁃week radiotherapy versus hypofractionated radiotherapy in patients older than 60 years with glioblastoma:the Nordic random⁃ised,phase 3 trial[J].Lancet Oncol,2012,13(9):916-926.
[36]Wick W,Platten M,Meisner C,et al.Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly:the NOA⁃08 randomised,phase 3 trial[J].Lancet Oncol,2012,13(7):707-715.
[37]Wan PT,Garnett MJ,Roe SM,et al.Mechanism of activation of the RAF⁃ERK Signaling pathway byoncogenic mutations of BRAF[J].Cell,2004,116(6):855-867.
[38]Korshunov A,Meyer J,Capper D,et al.Combined molecular analysis of BRAF and IDH1 distinguishes pilocytic astrocytoma from diffuse astrocytoma[J].Acta Neuropathol,2009,118(3):401-405.
[39]Schindler G,Capper D,Meyer J,et al.Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma,ganglioglioma and extra⁃cerebellar pilocytic astrocytoma[J].Acta Neuropathol,2011,121(3):397-405.
[40]Chapman PB,Hauschild A,Robert C,et al.Improved survival with vemurafenib in melanoma with BRAF V600E mutation[J].N Engl J Med,2011,364:2507-2516.
[41]Chamberlain MC.Recurrent ganglioglioma in adults treated with BRAF inhibitors[J].CNS Oncol,2016,5(1):27-29.
[42]Rush S,Foreman N,Liu A.Brainstem ganglioglioma successfully treated with vemurafenib[J].J Clin Oncol,2013,31(10):e159-e160.
[43]Kleinschmidt⁃DeMasters BK,Aisner DL,Foreman NK.BRAF VE1 immunoreactivity patterns in epithelioid glioblastomas positive for BRAF V600E mutation[J].Am J Surg Pathol,2015,39(4):528-540.
[44]Robinson GW,Orr BA,Gajjar A.Complete clinical regression of a BRAF V600E⁃mutant pediatric glioblastoma multiforme after BRAF inhibitor therapy[J].BMC Cancer,2014,14:258.
[45]Cancer Genome Atlas Research Network,Brat DJ,Verhaak RG,et al.Comprehensive,integrative genomic analysis of diffuse lower⁃grade gliomas[J].N Engl J Med,2015,372(26):2481-2498.
[46]Brennan CW,Verhaak RGW,McKenna A,et al.The somatic genomic landscape of glioblastoma[J].Cell,2013,155(2):462-477.
Progress on molecular diagnosis in glioma classification
MA Hui1,WEI Ming⁃Hai2,HU Zeng⁃Chun2
1Pharmaceutical Department,2Neurosurgeons,the Second Affiliated Hospital of Dalian Medical University,Dalian 116033,China
The treatment and prognosis of glioma is still a huge challenge.Recently,with the discovery of key genes and molecular markers,WHO has updated the diagnosis and classification criteria of glioma.These updates,such as IDH,1p/19q,K27M,and so on,require a systematic review of past case data and rethinking and designing relevant clinical and basic research.In this paper,we summarize the latest gene and molecular markers of glioma classification,and a selection of corresponding clinical treatment.
glioma;molecular diagnosis;classification;treat⁃ment;prognosis
R739.41
A
2095⁃6894(2017)07⁃64⁃04
2017-04-11;接受日期:2017-04-27
马 辉.硕士.E⁃mail:mahui⁃82@163.com
胡增春.副主任医师,硕导.E⁃mail:huzengchun@hotmail.com