多基因联合检测在判断膀胱癌预后价值中的意义

摘要: 目的探讨膀胱癌低级别与高级别两条通路在发生发展过程中的分子变化规律。方法应用PCR或低变性温度共扩增PCR(COLD-PCR)与Sanger直接测序法检测88例膀胱癌及10例对照组织中fgfr3、p53与h-ras基因突变状况, 以及MRP-1/CD9 mRNA表达水平及各基因与肿瘤复发之间的关系。Logistic回归及相关性分析比较各基因在肿瘤复发中的意义及相互关系。结果癌组织中p53突变率随病理分期及分级的增加而增加, MT-p53患者复发率高于WT-p53;fgfr3突变率则与之相反; 低级别病理分期及分级膀胱癌以MT-fgfr3/WT-p53基因型为主, 高级别病理分期及分级膀胱癌以WT-fgfr3/MT-p53基因型为主。h-ras突变率为11.4%(10/88), 主要分布于低级别病理分期及分级膀胱癌中。MRP-1/CD9 mRNA表达随病理分期及分级的增高而降低, 其表达与p53突变率呈负相关, 与fgfr3突变率呈正相关。在膀胱癌患者中WT-fgfr3复发危险为MT-fgfr3的3.88倍, MT-p53复发危险为WT-p53的4.53倍。结论低级别病理分期及分级膀胱癌发生发展中以fgfr3及h-ras基因突变为主, 高级别病理分期及分级肿瘤以p53基因突变、MRP-1/CD9 mRNA表达降低为主。fgfr3与p53突变是预测膀胱癌复发的有力指标, 在膀胱癌发生发展中分别代表不同的遗传学通路, 但低级别与高级别两通路有互相重叠。
- 膀胱癌 /
- 基因突变 /
- 预后 /
- 低变性温度共扩增PCR
Abstract: Objective To investigate the molecular changes in bladder urothelial carcinoma via different pathways. Methods Polymerase-chain reaction (PCR) or coamplification at low denaturation temperature-PCR and Sanger direct sequencing were performed to detect the status of fgfr3, p53, and h-ras gene mutations in 88 tissue samples of human bladder cancer and 10 normal control tissues. The relative mRNA expression levels of motility-related protein-1 (MRP-1)/CD9 and the relationship between genes and tumor recurrence were also determined. Logistic regression and relative analyses were conducted to compare the significance and interrelation of genes among tumor recurrences. Results The mutation rate of p53 increased as pathological grades and stages increased. Recurrence rate was higher in patients with MT-p53 genotype than in patients with WT-p53 genotype. Conversely, the mutation rate of fgfr3 gene decreased as pathological grades and stages increased. Recurrence rate was also higher in patients with WT-fgfr3 genotype than in patients with MT-fgfr3 genotype. In low-grade and early stage tumors, MT-fgfr3/WT-p53 was the most prevalent genotype; in high-grade and late stage tumors, WT-fgfr3/MT-p53 was the most prevalent genotype. The mutations of h-ras were mainly observed in low-grade tumors in early stages. Moreover, the relative mRNA levels of MRP-1/CD9 decreased as pathological grades and stages increased. The mRNA levels of MRP-1/CD9 were negatively correlated with p53 mutations and positively correlated with fgfr3 mutations. Logistic regression analysis results showed that patients with WT-fgfr3 genotypes exhibited 3.88 times higher relative risk of tumor recurrence than those with MT-fgfr3 genotypes; by contrast, patients with MT-p53 genotypes exhibited 4.53 times higher relative risk of tumor recurrence than those with WT-p53 genotypes. Conclusion Fgfr3 and h-ras gene mutations may play important roles in tumorigenesis of low-grade and early stage bladder cancer. p53 gene mutation and mRNA levels of MRP-1/CD9 may be implicated in the tumorigenesis of high-grade tumors in late stage of bladder cancer. In general, the two variants of urothelial carcinoma exhibit distinct genetic defects. fgfr3 gene mutation revealed a pathway of favorable prognosis, and p53 gene mutation demonstrated a pathway associated with poor prognosis.
- bladder cancer /
- gene mutation /
- prognosis /
- co-amplification at lower denaturation temperature-PCR

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图 1 p53与fgfr3基因突变测序图谱

Figure 1. Sequence map of p53 and fgfr3 gene mutations

A. CTG-CCG, the mutational site of codon 265 in p53 gene; B. Normal control of codon 265 in p53 gene. C. Heterozygous mutation of codon 249 in fgfr3 gene; D. Normal control of codon 249 in fgfr3 gene

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图 2 h-ras基因突变测序图谱

Figure 2. Sequence map of h-ras gene mutation

A. Heterozygous mutation of codon 12 in h-ras gene; B. Normal control of codon 12 in h-ras gene

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图 3 膀胱癌不同病理分级中p53与fgfr3基因突变分布类型

Figure 3. Distribution pattern of p53 and fgfr3 gene mutations in different pathological grades

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图 4 膀胱癌不同病理分期中p53与fgfr3基因突变分布类型

Figure 4. Distribution pattern of p53 and fgfr3 gene mutations in different pathological stages

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表 1 fgfr3、p53、h-ras、MRP-1/CD9及内参基因以mRNA为模板的引物序列

Table 1. Primer sequences using mRNA as templates of fgfr3, p53, h-ras, MRP-1/CD9, and reference genes

表 2 膀胱癌复发的多因素非条件Logistic回归分析

Table 2. Multifactor logistic regression analysis of patients with bladder cancer recurrencce

参考文献(15)

[1]	Mitra AP, Hansel DE, Cote RJ. Prognostic value of cell-cycle regulation biomarkers in bladder cancer[J]. Semin Oncol, 2012, 39(5): 524-533. doi: 10.1053/j.seminoncol.2012.08.008
[2]	Wang AX, Chang JW, Li CY, et al. H-ras mutation detection in bladder cancer by COLD-PCR analysis and direct sequencing[J]. Urol Int, 2012, 88(3):350-357. doi: 10.1159/000336132
[3]	赵丽,张淑敏,赵园园,等.高恶性膀胱移行细胞癌的蛋白质组学研究及差异蛋白mortalin的表达验证[J].中国肿瘤临床,2011,38(4):193-196. doi: 10.3969/j.issn.1000-8179.2011.04.004 Zhao L, Zhang SM, Zhao YY, et al. Proteomic analysis of highly malignant bladder transitional cell carcinoma and verification of mortalin[J]. Chin J Clin Oncol, 2011, 38(4):193-196. doi: 10.3969/j.issn.1000-8179.2011.04.004
[4]	Netto GJ. Molecular genetics and genomics progress in urothelial bladder cancer[J]. Semin Diagn Pathol, 2013, 30(4):313-320. doi: 10.1053/j.semdp.2013.11.005
[5]	Li J, Wang L, Mamon H, et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing[J]. Nat Med, 2008, 14(5):579-584. doi: 10.1038/nm1708
[6]	Pollard C, Smith SC, Theodorescu D. Molecular genesis of nonmuscle-invasive urothelial carcinoma (NMIUC) [J]. Expert Rev Mol Med, 2010, 12:e10. doi: 10.1017/S1462399410001407
[7]	van Rhijn BW, van der Kwast TH, Vis AN, et al. FGFR3 and p53 characterize alternative genetic pathways in the pathogenesis of urothelial cell carcinoma[J]. Cancer Res, 2004, 64(6):1911-1914. doi: 10.1158/0008-5472.CAN-03-2421
[8]	Ben Abdelkrim S, Rammeh S, Ziadi S, et al. Expression of topoisomerase II alpha, ki67, and p53 in primary non-muscle-invasive urothelial bladder carcinoma[J]. J Immunoassay Immunochem, 2014, 35(4):358-367. doi: 10.1080/15321819.2014.899254
[9]	Dodurga Y, Tataroglu C, Kesen Z, et al. Incidence of fibroblast growth factor receptor 3 gene (FGFR3) A248C, S249C, G372C, and T375C mutations in bladder cancer[J]. Genet Mol Res, 2011, 10(1):86-95. doi: 10.4238/vol10-1gmr923
[10]	Cappellen D, De Oliveira C, Ricol D, et al. Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas[J]. Nat Genet, 1999, 23(1):18-20. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=667332060075fd5f1d4c3b1189514c0e
[11]	Liu X, Zhang W, Geng D, et al. Clinical significance of fibroblast growth factor receptor-3 mutations in bladder cancer: a systematic review and meta-analysis[J]. Genet Mol Res, 2014, 13(1):1109-1120. doi: 10.4238/2014.February.20.12
[12]	Bakkar AA, Wallerand H, Radvanyi F, et al. FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder[J]. Cancer Res, 2003, 63(23):8108-8112.
[13]	Ouerhani S, Elgaaied AB. The mutational spectrum of HRAS, KRAS, NRAS and FGFR3 genes in bladder cancer[J]. Cancer Biomark, 2011, 10(6):259-266.
[14]	Li J, Wang L, Jänne PA, et al. Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR[J]. Clin Chem, 2009, 55(4):748-756. doi: 10.1373/clinchem.2008.113381
[15]	Mhawech P, Herrmann F, Coassin M, et al. Motility-related protein 1 (MRP-1/CD9) expression in urothelial bladder carcinoma and its relation to tumor recurrence and progression[J]. Cancer, 2003, 98(8):1649-1657. doi: 10.1002/cncr.11698