CDC5L表达对神经母细胞瘤临床及预后评估的意义
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摘要
目的探讨CDC5L在神经母细胞瘤(neuroblastoma,NB)中的表达及对患儿临床和预后的影响。方法免疫组化(immunohistochemistry,IHC)及蛋白免疫印迹(Western blot,WB)检测CDC5L在神经母细胞瘤患儿肿瘤组织中的表达,根据患儿性别、年龄、是否有骨髓浸润、肿瘤临床分期(INSS)、危险度分级、肿瘤病理诊断、Shimada病理分类及NSE、LDH是否正常进行分组,比较各组中CDC5L的表达差异,采用Kaplan-Meier和Cox回归分析CDC5L高表达组和低表达组对神经母细胞瘤生存的影响。结果 IHC显示,62例神经母细胞瘤肿瘤标本中,CDC5L高表达及低表达各31例(50%),CDC5L主要在细胞核内表达; IHC及WB均显示CDC5L在神经母细胞瘤中的表达高于节细胞神经母细胞瘤(ganglioneuroblastoma,GNB),NB和GNB均起源于原始神经嵴细胞,可于肾上腺或沿脊柱旁交感神经节分布;不同之处在于NB较GNB分化差,恶性程度更高。进一步分析CDC5L在NB患儿临床和病理特征之间的表达差异发现肿瘤骨髓浸润组较无肿瘤浸润组CDC5L高表达的比例更高,差异有统计学意义(χ~2=5.833,P=0.016);CDC5L在肿瘤临床分期Ⅲ～Ⅳ中的高表达比例高于Ⅰ～Ⅱ和Ⅳ-S期,差异有统计学意义(χ~2=11.328,P=0.001);CDC5L高表达的比例在MYCN扩增组高于MYCN非扩增组(P=0.038);危险度分级中,高危患儿中CDC5L表达增高的比例较中危和低危患儿高(χ~2=9.378,P=0.002);Shimada病理分类中,不良组织学类型较良好组织学类型CDC5L表达增高的比例更高(χ~2=7.839,P=0.005);NSE升高组较NSE正常组CDC5L表达增高的比例更高(P=0.035);LDH≥587 U/L较LDH<587 U/L有更高比例的CDC5L表达增高,差异有统计学意义(χ~2=5.547,P=0.019)。Kaplan-Meier生存分析提示CDC5L高表达组3年存活率(48.8%vs. 88.0%)低于CDC5L低表达组,差异有统计学意义(P=0.002)。单因素Cox回归分析发现CDC5L高表达(HR=4.734,95%CI:1.590～14.091)、临床分期为Ⅲ和Ⅳ期(HR=7.444,95%CI:2.187～25.339)、高危状态(HR=2.749,95%CI:1.454～5.194)、Shimada病理UFH型(HR=4.098,95%CI:1.493～11.250)均是NB患儿预后的危险因素。但是进一步多因素Cox回归分析发现上述因素均没有统计学差异(P>0.05)结论 CDC5L的表达和已知的患儿预后危险因素、不良病理类型及肿瘤标志物升高有较好的一致性,且生存分析也提示CDC5L高表达组较低表达组差,因此CDC5L可作为潜在的提示神经母细胞瘤患儿具有不良预后、不良病理类型和肿瘤标志物升高的分子标志物。
Objective To explore the expression of CDC5 L and its influence on clinicopathological characteristics in children of neuroblastoma(NB). Methods The expression of CDC5 L was detected by immunohistochemistry(IHC) and Western blot(WB) in 62 NB specimens. They were divided into several groups according to gender,age,metastasis of bone marrow,tumor clinical stage(INSS),risk rating,pathological diagnosis,Shimada pathological classification and the results of lactate dehydrogenase(LDH) and neuron specific enolase(NSE). Then the difference of CDC5 L expression between groups was analyzed by chi-square test. We also analyzed the overall survival rate between low and high CDC5 L expression groups in 50 NB patients with complete follow-up data using Kaplan-Meier and COX regression. Results Immunohistochemical staining showed that CDC5 L was predominantly located at nucleus of NB cells. Both high and low expressions of CDC5 L were detected in 31/62 NB patients(50%). Both IHC and WB showed that CDC5 L was more strongly expressed in NB than ganglioneuroblastoma(GNB). Both NB and GNB were derived from neural crest cells,might arise in adrenal medulla or anywhere along paraspinal sympathetic ganglia while NB was less differentiated and more malignant; We further analyzed the relationship between CDC5 L expression and other clinicopathological characteristics. It was found that CDC5 L expression level was higher in bone marrow infiltration group than that in non-infiltration group(χ~2=5.833,P=0.016). The expression of CDC5 L was higher in stage Ⅲ-Ⅳ cases than that in stage Ⅰ-Ⅱ/Ⅳ-S cases(χ~2=11.328,P=0.001). Overexpression of CDC5 L was usually accompanied by a frequent amplification of MYCN and amplification status of MYCN was different between high and low expression groups of CDC5 L(P=0.038); CDC5 L was more abundant in high-risk group than in low/intermediate-risk group(χ~2=9.378,P=0.002). According to the pathological classification of Shimada,unfavorable histologic category(UFH) cases had a higher expression of CDC5 L than favorable histologic category cases(χ~2=7.839,P=0.005). The expression of CDC5 L was higher in NSE-elevated group than that in NSE-normal group(P=0.035). Cases with LDH≥587 U/L had a higher expression of CDC5 L(χ~2=5.547,P=0.019). Both Kaplan-Meier(P=0.002) and COX regression(HR=4.734,95%CI:1.590-14.091) showed that overexpression of CDC5 L had a poor clinical prognosis. In addition,higher clinical stage(HR=7.444,95%CI:2.187-25.339),high-risk state(HR=2.749,95%CI:1.454-5.194) and pathological UFH type(HR=4.098,95%CI:1.493-11.250) were associated with worse clinical outcomes. However,when the above factors were further analyzed by COX multivariate regression,no statistical difference was observed. Conclusion A high expression of CDC5 L is highly consistent with prognostic risk factors,adverse pathological types and elevated tumor markers. Kaplan-Meier curves also shows that low CDC5 L group has longer survival time than high CDC5 L group. Therefore CDC5 L may be used as a potential molecular marker of poor prognosis,poor pathological type and elevation of tumor makers in NB children.
Objective To explore the expression of CDC5 L and its influence on clinicopathological characteristics in children of neuroblastoma(NB). Methods The expression of CDC5 L was detected by immunohistochemistry(IHC) and Western blot(WB) in 62 NB specimens. They were divided into several groups according to gender,age,metastasis of bone marrow,tumor clinical stage(INSS),risk rating,pathological diagnosis,Shimada pathological classification and the results of lactate dehydrogenase(LDH) and neuron specific enolase(NSE). Then the difference of CDC5 L expression between groups was analyzed by chi-square test. We also analyzed the overall survival rate between low and high CDC5 L expression groups in 50 NB patients with complete follow-up data using Kaplan-Meier and COX regression. Results Immunohistochemical staining showed that CDC5 L was predominantly located at nucleus of NB cells. Both high and low expressions of CDC5 L were detected in 31/62 NB patients(50%). Both IHC and WB showed that CDC5 L was more strongly expressed in NB than ganglioneuroblastoma(GNB). Both NB and GNB were derived from neural crest cells,might arise in adrenal medulla or anywhere along paraspinal sympathetic ganglia while NB was less differentiated and more malignant; We further analyzed the relationship between CDC5 L expression and other clinicopathological characteristics. It was found that CDC5 L expression level was higher in bone marrow infiltration group than that in non-infiltration group(χ~2=5.833,P=0.016). The expression of CDC5 L was higher in stage Ⅲ-Ⅳ cases than that in stage Ⅰ-Ⅱ/Ⅳ-S cases(χ~2=11.328,P=0.001). Overexpression of CDC5 L was usually accompanied by a frequent amplification of MYCN and amplification status of MYCN was different between high and low expression groups of CDC5 L(P=0.038); CDC5 L was more abundant in high-risk group than in low/intermediate-risk group(χ~2=9.378,P=0.002). According to the pathological classification of Shimada,unfavorable histologic category(UFH) cases had a higher expression of CDC5 L than favorable histologic category cases(χ~2=7.839,P=0.005). The expression of CDC5 L was higher in NSE-elevated group than that in NSE-normal group(P=0.035). Cases with LDH≥587 U/L had a higher expression of CDC5 L(χ~2=5.547,P=0.019). Both Kaplan-Meier(P=0.002) and COX regression(HR=4.734,95%CI:1.590-14.091) showed that overexpression of CDC5 L had a poor clinical prognosis. In addition,higher clinical stage(HR=7.444,95%CI:2.187-25.339),high-risk state(HR=2.749,95%CI:1.454-5.194) and pathological UFH type(HR=4.098,95%CI:1.493-11.250) were associated with worse clinical outcomes. However,when the above factors were further analyzed by COX multivariate regression,no statistical difference was observed. Conclusion A high expression of CDC5 L is highly consistent with prognostic risk factors,adverse pathological types and elevated tumor markers. Kaplan-Meier curves also shows that low CDC5 L group has longer survival time than high CDC5 L group. Therefore CDC5 L may be used as a potential molecular marker of poor prognosis,poor pathological type and elevation of tumor makers in NB children.
引文
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11 中国抗癌协会小儿肿瘤专业委员会.儿童神经母细胞瘤诊疗专家共识[J].中华小儿外科杂志,2015,36(1):3 — 7.DOI:10.3760/cma.j.issn.0253 — 3006.2015.01.00.Chinese Children Cancer Group.Expert consensus on diagnosis and treatment of neuroblastoma in Children[J].Chin J Pediatr Surg,2015,36(1):3 — 7.DOI:10.3760/cma.j.issn.0253 — 3006.2015.01.00.
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14 董岿然.我国儿童胚胎性恶性实体肿瘤的研究和治疗进展[J].临床小儿外科杂志,2017,16(5):417 — 421.DOI:DOI:10.3969/j.issn.1671 — 6353.2017.05.001.Dong KR.Diagnosis and treatment of embryonic malignant tumors in children in China [J].J Clin Ped Sur,2017,16(5):417 — 421.DOI:10.3969/j.issn.1671 — 6353.2017.05.001.
15 李斯文,王珊,杨超,等.药物相关性分子靶标检测在儿童恶性实体肿瘤个体化治疗中的初步研究[J].临床小儿外科杂志,2017,16(4):341 — 346.DOI:10.3969j.issn.1671 — 6353.2017.004.008.Li SW,Wang S,Yang C,et al.Preliminary identification of drug-related genomic markers for individualized therapy in children with malignant solid tumors[J].J Clin Ped Sur,2017,16(4):341 — 346.DOI:10.3969/j.issn.1671 — 6353.2017.004.008.
16 Qiu H,Zhang X,Ni W,et al.Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma[J].Dig Dis Sci,2016,61(3):795 — 805.DOI:10.1007/s10620 — 015 — 3937 — 9.
17 Chao WC,Kulkarni K,Zhang Z,et al.Structure of the mitotic checkpoint complex[J].Nature,2012,484(7393):208 — 213.DOI:10.1038/nature10896.
18 Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646 — 674.DOI:10.1016/j.cell.2011.02.013.
19 Qiu H,Zhang X,Ni W,et al.Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma[J].Dig Dis Sci ,2016,61(3):795 — 805.DOI:10.1007/s10620 — 015 — 3937 — 9.
20 Chen W,Li Z,Yan W,et al.Expression of CDC5L is associated with tumor progression in gliomas[J].Tumour Biol,2016,37(3):4093 — 4103.DOI:10.1007/s13277 — 015 — 4088 — 5.
21 Mu R,Wang YB,Wu M,et al.Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe[J].Cell Death Dis,2014,5(3):e1151.DOI:10.1038/cddis.2014.117.
22 Mohammadi M,Goudarzi PK,Rahmani O,et al.Evaluation of gene expression level of CDC5L and MACC1 in poor prognosis and progression of osteosarcoma[J].Tumor Biology,2016,37(6):8153 — 8157.DOI:10.1007/s13277 — 015 — 4726 — y.
23 Brodeur GM,Bishop JM.Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage[J].Science,1984,224(4653):1121.DOI:10.1126/science.6719137.
24 Seeger RC,Brodeur GM,Sather H,et al.Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas[J].N Engl J Med,1985,313(18):1111 — 1116.DOI:10.1056/NEJM198510313131802.
25 Zeid R,Lawlor MA,Poon E,et al.Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma[J].Nat Genet,2018,50(4):515 — 523.DOI:10.1038/s41588 — 018 — 0044 — 9.
26 Kang JH,Rychahou PG,Ishola TA,et al.MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells[J].Biochem Biophys Res Commun,2006,351(1):192 — 197.DOI:10.1016/j.bbrc.2006.10.020.
27 Tonelli R,Purgato S,Camerin C,et al.Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis[J].Mol Cancer Ther,2005,4(5):779 — 786.DOI:10.1158/1535 — 7163.MCT — 04 — 0213.
28 Dubois SG,Kalika Y,Lukens JN,et al.Metastatic sites in stage IV and IVS neuroblastoma correlate with age,tumor biology,and survival[J].J Pediatr Hematol Oncol,1999,21(3):181 — 189.DOI:10.1097/00043426 — 199905000 — 00005.
29 De BB,Gambini C,Haupt R,et al.Retrospective study of childhood ganglioneuroma[J].J Clin Oncol,2008,26(10):1710 — 1716.DOI:10.1200/JCO.2006.08.8799.
30 Cohen MD.International criteria for neuroblastoma diagnosis,staging,and response to treatment[J].J Clin Oncol,1994,12(9):1991 — 1993.DOI:10.1200/JCO.1994.12.9.1991.
31 Shimada H,Ambros IM,Dehner LP,et al.The International Neuroblastoma Pathology Classification (the Shimada system)[J].Cancer,1999,86(2):364 — 372.DOI:10.1002/(sici)1097 — 0142(19990715)86:2<364:aid — cncr21>3.0.co;2 — 7.
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2 Jie Y,Lan W,Zhu JM,et al.Prp19 facilitates invasion of hepatocellular carcinoma via p38 mitogen-activated protein kinase/Twist1 pathway[J].Oncotarget,2016,7(16):21939 — 21951.DOI:10.18632/oncotarget.7877.
3 Huang R,Xue R,Qu D,et al.Prp19 Arrests Cell Cycle via Cdc5L in Hepatocellular Carcinoma Cells[J].Int J Mol Sci,2017,18(4):778.DOI:10.3390/ijms18040778.
4 Lu S,Wang R,Cai C,et al.Small Kinetochore Associated Protein (SKAP) Promotes UV-Induced Cell Apoptosis through Negatively Regulating Pre-mRNA Processing Factor 19 (Prp19)[J].PLoS One,2014,9(4):e92712.DOI:10.1371/journal.pone.0092712.
5 Bernstein HS,Coughlin SR.Pombe Cdc5-related protein.A putative human transcription factor implicated in mitogen-activated signaling[J].J Biol Chem,1997,272(9):5833 — 5837.DOI:10.1074/jbc.272.9.5833.
6 Zhang N,Kaur R,Akhter S,et al.Cdc5L interacts with ATR and is required for the S-phase cell-cycle checkpoint[J].Embo Reports,2009,10(9):1029.DOI:10.1038/embor.2009.122.
7 Boudrez A,Beullens M,Groenen P,et al.NIPP1-mediated interaction of protein phosphatase-1 with CDC5L,a regulator of pre-mRNA splicing and mitotic entry[J].J Biol Chem,2000,275(33):25411 — 25417.DOI:10.1074/jbc.M001676200.
8 Sadikovic B,Thorner P,Chilton-Macneill S,et al.Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy[J].BMC Cancer,2010,10(1):1 — 9.DOI:10.1186/1471 — 2407 — 10 — 202.
9 Martin JW,Chiltonmacneill S,Koti M,et al.Digital Expression Profiling Identifies RUNX2,CDC5L,MDM2,RECQL4,and CDK4 as Potential Predictive Biomarkers for Neo-Adjuvant Chemotherapy Response in Paediatric Osteosarcoma[J].PLoS One,2014,9(5):e95843.DOI:10.1371/journal.pone.0095843.
10 Cohn SL,Pearson AD,London WB,et al.The International Neuroblastoma Risk Group (INRG) classification system:an INRG Task Force report[J].J Clin Oncol,2009,27(2):289 — 297.DOI:10.1200/JCO.2008.16.6785.
11 中国抗癌协会小儿肿瘤专业委员会.儿童神经母细胞瘤诊疗专家共识[J].中华小儿外科杂志,2015,36(1):3 — 7.DOI:10.3760/cma.j.issn.0253 — 3006.2015.01.00.Chinese Children Cancer Group.Expert consensus on diagnosis and treatment of neuroblastoma in Children[J].Chin J Pediatr Surg,2015,36(1):3 — 7.DOI:10.3760/cma.j.issn.0253 — 3006.2015.01.00.
12 Brodeur GM.Neuroblastoma:biological insights into a clinical enigma[J].Nature Reviews Cancer,2003,3(3):203 — 216.DOI:10.1038/nrc1014.
13 Maris JM,Hogarty MD,Bagatell R,et al.Neuroblastoma[J].Lancet,2007,369(9579):2106 — 2120.DOI:10.1016/S0140 — 6736(07)60983 — 0.
14 董岿然.我国儿童胚胎性恶性实体肿瘤的研究和治疗进展[J].临床小儿外科杂志,2017,16(5):417 — 421.DOI:DOI:10.3969/j.issn.1671 — 6353.2017.05.001.Dong KR.Diagnosis and treatment of embryonic malignant tumors in children in China [J].J Clin Ped Sur,2017,16(5):417 — 421.DOI:10.3969/j.issn.1671 — 6353.2017.05.001.
15 李斯文,王珊,杨超,等.药物相关性分子靶标检测在儿童恶性实体肿瘤个体化治疗中的初步研究[J].临床小儿外科杂志,2017,16(4):341 — 346.DOI:10.3969j.issn.1671 — 6353.2017.004.008.Li SW,Wang S,Yang C,et al.Preliminary identification of drug-related genomic markers for individualized therapy in children with malignant solid tumors[J].J Clin Ped Sur,2017,16(4):341 — 346.DOI:10.3969/j.issn.1671 — 6353.2017.004.008.
16 Qiu H,Zhang X,Ni W,et al.Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma[J].Dig Dis Sci,2016,61(3):795 — 805.DOI:10.1007/s10620 — 015 — 3937 — 9.
17 Chao WC,Kulkarni K,Zhang Z,et al.Structure of the mitotic checkpoint complex[J].Nature,2012,484(7393):208 — 213.DOI:10.1038/nature10896.
18 Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646 — 674.DOI:10.1016/j.cell.2011.02.013.
19 Qiu H,Zhang X,Ni W,et al.Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma[J].Dig Dis Sci ,2016,61(3):795 — 805.DOI:10.1007/s10620 — 015 — 3937 — 9.
20 Chen W,Li Z,Yan W,et al.Expression of CDC5L is associated with tumor progression in gliomas[J].Tumour Biol,2016,37(3):4093 — 4103.DOI:10.1007/s13277 — 015 — 4088 — 5.
21 Mu R,Wang YB,Wu M,et al.Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe[J].Cell Death Dis,2014,5(3):e1151.DOI:10.1038/cddis.2014.117.
22 Mohammadi M,Goudarzi PK,Rahmani O,et al.Evaluation of gene expression level of CDC5L and MACC1 in poor prognosis and progression of osteosarcoma[J].Tumor Biology,2016,37(6):8153 — 8157.DOI:10.1007/s13277 — 015 — 4726 — y.
23 Brodeur GM,Bishop JM.Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage[J].Science,1984,224(4653):1121.DOI:10.1126/science.6719137.
24 Seeger RC,Brodeur GM,Sather H,et al.Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas[J].N Engl J Med,1985,313(18):1111 — 1116.DOI:10.1056/NEJM198510313131802.
25 Zeid R,Lawlor MA,Poon E,et al.Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma[J].Nat Genet,2018,50(4):515 — 523.DOI:10.1038/s41588 — 018 — 0044 — 9.
26 Kang JH,Rychahou PG,Ishola TA,et al.MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells[J].Biochem Biophys Res Commun,2006,351(1):192 — 197.DOI:10.1016/j.bbrc.2006.10.020.
27 Tonelli R,Purgato S,Camerin C,et al.Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis[J].Mol Cancer Ther,2005,4(5):779 — 786.DOI:10.1158/1535 — 7163.MCT — 04 — 0213.
28 Dubois SG,Kalika Y,Lukens JN,et al.Metastatic sites in stage IV and IVS neuroblastoma correlate with age,tumor biology,and survival[J].J Pediatr Hematol Oncol,1999,21(3):181 — 189.DOI:10.1097/00043426 — 199905000 — 00005.
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