结直肠癌组织中CD44v3,v6蛋白的表达意义
复旦大学医学院附属肿瘤医院病理科 上海市 200032
蔡崎，女，1972-01-16生，辽宁省大连市人，汉族. 1994年上海医科大学医学系本科毕业. 现复旦大学医学院(原上海医科大学)附属肿瘤医院肿瘤病理专业博士在读. 研究方向：胃肠道肿瘤的病理学和肿瘤转移.
项目负责人 蔡崎，200032，上海市零陵路399号，复旦大学医学院附属肿瘤医院病理科.
Department of Pathology, Cancer Hospital/Cancer Institute of Medical Center of Fudan University, Shanghai 200032, China
Correspondence to Qi Cai, Department of Pathology, Cancer Hospital/Cancer Institute of Medical Center of Fudan University,Shanghai 200032, China
Tel. 0086-21-64175590 Ext.3357
Email. caiqi@baoscape.com
Received 2000-05-18 Accepted 2000-06-02
Expression of CD44 v3 and v6 proteins in human colorectal carcinoma and its relevance with prognosis
Qi Cai, Hong-Fen Lu, Meng-Hong Sun, Xiang Du, Yue-Zhen Fan and Da-Ren Shi
Abstract
AIM To evaluate the expression of CD44v3 and v6 protein in colorectal carcinoma and its prognostic significance.
METHODS One hundred and twenty-one cases of formalin-fixed paraffin-embedded colorectal carcinoma were retrospectively analyzed using EnvisionTM immunohistochemical method with the monoclonal antibody CD44v3 and v6. The median following-up time was 67.77 months and the prognostic value of the CD44v3 and CD44v6 was assessed using univariate and multivariate survival analysis.
RESULTS The positive rates of CD44v3 and v6 protein were 60.3% and 57.9%, respectively. There was significant correlation between CD44v3 immunoreactivity and tumor location,lymph node metastasis, distant metastasis and Duke＇s stage (P<0.05, Spearman correlation test). Significant correlation between CD44v6 immunoreactivity and patients＇ gender, lymph node metastasis, distant metastasis, Duke＇s stage was also noticed (P<0.05, Spearman correlation test). The 5-year survival rates were 81.25% and 60.27% in CD44v3 negative cases and CD44v3 positive ones, respectively. As CD44v6, the 5-year survival rates were 80.39% and 60.00% in CD44v6 negative cases and CD44v6 positive ones, respectively; the differences between the two groups of patients were significant (P<0.05, Log-rank test). In multivariate analysis using the Cox regression model, CD44v3 expression emerges as an independent prognostic indicator.
CONCLUSION CD44v3 and v6 might play some important roles in metastasis of colorectal carcinoma, and CD44v3 expression might be a new useful independent prognostic marker of colorectal carcinoma.
Subject headings colorectal neoplasms; cell adhesion molecules; CD44v3; CD44v6; neoplasm metastasis; immunohistochemistry
Cai Q, Lu HF, Sun MH, Du X, Fan YZ, Shi DR. Expression of CD44 v3 and v6 proteins in human colorectal carcinoma and its relevance with prognosis. Shijie Huaren Xiaohua Zazhi, 2000;8(11):1255-1258
摘要
目的 研究CD44v3, v6蛋白在结直肠癌组织中的表达及其预后意义.
方法 应用EnvisionTM 免疫组化法，回顾性分析121例结直肠癌石蜡组织CD44v3, v6的表达. 患者中位随访时间为67.77mo.
结果 CD44v3, v6在结直肠癌中的阳性率分别为60.3%和57.9%.CD44v3的阳性表达与患者肿瘤部位，淋巴结转移状况、远处转移与否、临床分期密切相关(P<0.05，Spearman等级相关检验). CD44v6的阳性表达与患者性别、淋巴结转移状况、远处转移与否、临床分期密切相关(P<0.05，Spearman等级相关检验). Kaplan-Meier生存曲线，Log-rank检验单因素生存分析结果显示，CD44v3阴性、阳性患者的五年生存率分别为81.25%, 60.27%，两者之间存在显著性差异(P=0.035)；CD44v6阴性、阳性患者的5a生存率分别为80.39%, 60.00%，两者之间亦存在显著性差异(P=0.0299). Cox模型多因素生存分析结果显示，CD44v3表达是影响结直肠癌预后的独立因素.
结论 CD44v3, v6蛋白与结直肠癌转移、预后相关，CD44v3是影响结直肠癌预后的独立因素.
主题词 结肠直肠肿瘤；细胞粘附分子；CD44v3；CD44v6；肿瘤转移；免疫组织化学
蔡崎, 陆洪芬, 孙孟红, 杜祥, 范月珍, 施达仁. 结直肠癌组织中CD44v3, v6蛋白的表达意义. 世界华人消化杂志，2000；8(11)：1255-1258
0 引言
结直肠癌是一种常见的消化道恶性肿瘤，近年来发病率不断增加. 以上海为例，上海市肿瘤研究所流行病学研究室统计资料显示：1996年结直肠癌发病率为69.9/10万，并有继续上升趋势[1]. 转移的发生是导致结直肠癌患者死亡的主要原因，是临床治疗的最大难题之一，影响着患者的预后. 对结直肠癌转移潜能、预后的有效评估非常重要，能够指导临床治疗方案的制定，不仅可使适宜的患者及时接受必要的辅助治疗，也可使某些患者避免承受不必要的放疗、化疗所致的副作用，从而提高患者的生存率和生存质量[2-4]. 近年来细胞粘附分子CD44,尤其CD44变异体(CD44v)与肿瘤转移的关系正日益受到人们的重视[5-24]. 然而结直肠癌CD44v方面研究报道尚少，且结果不一. 故本文采用免疫组化方法检测结直肠癌组织中CD44v3,v6蛋白的表达情况，并观察其与转移、预后的关系.
1 材料和方法
1.1 材料 我院1991-01/1994-04临床病理资料完整的结直肠癌121例(结肠癌50例，直肠癌71例). 其中男72例，女49例. 年龄22～88(平均55，中位57)岁. 高分化腺癌8例，中分化腺癌89例，低分化腺癌9例，粘液腺癌14例，印戒细胞癌1例. Duke＇s A期20例，Duke＇s B期31例，Duke＇s C 期58例，Duke＇s D期12例. 随访至1999-04，死亡42例，存活79例，存活时间1.50～100.27(平均59.75，中位67.77)mo. 所有病例术前均未经放疗和化疗，并由两位病理医生确诊. 另取40例距癌10cm以远处肠粘膜作对照研究. 抗CD44v3,抗CD44v6mAb购自Bender Medsystem 公司，工作稀释度分别为1∶40和1∶400. Envision反应液购自Dako公司.
1.2 方法 取存档蜡块制备4μm连续切片，二甲苯脱蜡、梯度酒精水化后置于pH=6.0的枸橼酸盐缓冲液中，微波法修复抗原：95℃, 5min×4次，EnvisionTM免疫组化法染色. DAB显色，苏木素复染，常规脱水、透明、封片，观察结果. 用TBS代替一抗作阴性对照，正常皮肤鳞状上皮作阳性对照. CD44v3阳性定位于胞质，CD44v6阳性定位于胞膜. 免疫组化结果采用半定量计分法判定. 即A.按阳性着色程度评分. 0分：无着色；1分：浅黄色；2分：棕黄色；3分：棕褐色. B.按阳性细胞所占比例评分. 0分：<5%；1分：5%～10%; 2分：11%～50%；3分：51%～80%；4分：>80%. 两者乘积：0分为阴性(-)；1～4分为弱阳性(+)；5～8分为中度阳性(++)；9～12分为强阳性(+++).
统计学处理 应用SPSS8.2软件spearman相关分析. 生存分析采用Kaplan-meier生存曲线、Log-rank单因素分析及Cox风险模型多因素分析.
2 结果
正常肠粘膜均不表达CD44v3, v6. 结直肠癌组织CD44v3表达阳性率为60.4%. 其中+者占43.0%，++者占14.9%，+++者占2.5%. CD44v6表达阳性率为57.9%. 其中+者占43.8%，++者占10.7%，+++者占3.3%. 且CD44v3与CD44v6的表达密切相关
(P<0.05，表1).
表1 CD44v6, v3表达的相关性
P=0.000, r=0.411, Spearman 相关分析.
2.1 CD44v3, v6表达与临床病理特征 免疫组化结果显示，CD44v3阳性表达与患者年龄、性别、大体类型、组织学类型、肿块最大径、脉管神经侵犯、肿瘤浸润深度均无显著相关(P>0.05)，而与肿瘤部位、淋巴结转移状况、有无远处转移及Duke＇s分期密切相关(P<0.05，表2). CD44v6阳性表达与患者年龄、部位、大体类型、组织学类型、肿块最大径、脉管神经侵犯、肿瘤浸润深度均无显著相关(P>0.05)，而与患者性别、淋巴结转移状况、有无远处转移及Duke＇s分期密切相关(P<0.05，表2).
表2 CD44v6, v3的表达与结直肠癌患者临床病理特征的关系
Spearman 相关分析.
2.2 CD44v3, v6表达与生存率 绘制Kaplan-Meier生存曲线，采用Log-rank单因素检验对121例结直肠癌患者的生存情况进行分析，结果显示：CD44v3阴性患者的五年生存率为81.25%，阳性患者的五年生存率为60.27%，二者之间存在显著性差异(P=0.0035，图1). CD44v6阴性、阳性患者的五年生存率分别为80.39%, 60.00%，二者之间亦存在显著性差异(P=0.0299，图2). 此外，在临床病理特征中，肿瘤浸润深度，淋巴结转移状况，有无远处转移，神经侵犯及Duke＇s分期与患者生存情况相关(P<0.05). 而年龄，性别，大体类型，组织学类型，部位，脉管侵犯，肿块最大径与预后无显著性相关(P>0.05). 应用Cox模型进行多因素分析显示：Duke＇s分期，神经侵犯，CD44v3是影响结直肠癌预后的独立因素(表3)，而CD44v6未进入最终模型.
表3 Cox回归多因素生存分析
图1 CD44v6表达与患者生存的关系(Log-rank test, P=0.0299).
图2 CD44v3表达与患者生存的关系(Log-rank test, P=0.0035).
3 讨论
恶性肿瘤细胞重要的生物学特征之一表现为细胞与细胞之间、细胞与细胞外基质之间粘附特性的异常，这种粘附特性的异常导致肿瘤细胞具有了从原发部位脱落并向周围组织侵袭及向远隔器官转移的能力. CD44正是一个近年来倍受关注，在细胞恶性转化过程中其结构和功能均发生显著变化而影响转化后肿瘤细胞侵袭转移行为的细胞表面粘附分子[17]. CD44作为具有高度异质性的单链跨膜糖蛋白，在体内分布极广泛，能与透明质酸等多种配体结合，参与细胞与细胞、细胞与基质之间的粘连. 其具有高度异质性，系编码该蛋白的基因在转录过程中的mRNA选择性拼接和蛋白质合成过程中的翻译后修饰不同所致. 主要有两种类型，标准型(CD44s)和变异型(CD44v)，后者又由于选择性拼接方式不同而包含有多种类型[25-28]. 近年来研究表明,CD44v可在不同水平被调节，参与肿瘤的侵袭和转移[6,25,26].
CD44v6作为较为公认的转移相关因子，最早由动物实验发现，将大鼠转移性胰腺癌细胞株(BSP73ASML)的CD44vDNA转染到非转移的BSP73AS癌细胞株中，发现后者发生了转移[29]，经测定CD44v4-7和CD44v6-7是BSP73ASML株中表达最强的两种CD44v[30]，而预先用抗CD44v抗体处理可能防止转移的发生[31]. 随后，不同学者用不同方法对不同肿瘤标本的不同CD44表达情况作了研究，发现大多数肿瘤如肠癌、胃癌、乳腺癌、肝癌等的CD44尤其CD44v表达增高，并在不同程度上与肿瘤的侵袭、转移相关[10-23]. 也有少数肿瘤如食管、头颈部及宫颈鳞癌则出现CD44v的负调节[24，32-34].
关于CD44v6与结直肠癌转移、预后关系的研究，不同学者结论不一. Wielenga et al[10]报道，含有v6序列的CD44表达通常限于肿瘤发展的后期，在转移性(Duke＇s C/D期)中的表达高于未发生转移者(Duke＇s A/B期). Mulder et al[35]研究亦显示CD44v6在肿瘤进展期有过度表达，CD44v6可作为一种独立的估计结直肠癌预后的标记物，并可用以指导Duke＇s B, C期结直肠癌治疗方案的制定. Wielenga et al[36]用免疫组化法(IHC)证实CD44v6在结直肠癌中的表达具有显著的预后意义. 而Gotley et al[37], Givehchian et al[38]研究显示CD44v与结直肠癌恶性进展和转移无关. Finke et al[39], Koretz et al[40], Coppola et al[41]认为CD44v6不能作为结直肠癌的预后指标. Weg-Remers et al[42]甚至研究显示CD44v6在转移性肠癌和转移灶中表达降低. 笔者对上述研究进行分析后认为，不同学者得出不同结论的原因可能如下：①肿瘤本身的异质性；取材的偏差. ②各实验室的操作不同，同时所用的试剂不同，各实验室所订的标准不一样. ③IHC方法所用的抗体特异性高低不一. ④研究方法不同，灵敏度存在差异. ⑤首先报道者可能会对所取得的结论过分估计其真正价值等等.
对CD44v3与肿瘤转移、预后关系的研究较少，Ropponen et al[43]认为其可能作为结直肠癌的预后指标. 而在对与乳腺癌进展、转移关系的研究中，Kalish et al[44]指出含有v3变异体的CD44v与乳腺癌的转移密切相关.
我们采用EnvisionTM免疫组化两步法，敏感度、特异性较高，背景清晰. 结果显示：与正常肠粘膜相比，CD44v6在结直肠癌中的表达显著升高. CD44v6的表达与患者年龄、性别、肿瘤大小等均无相关，而与淋巴结转移、远处转移密切相关，发生淋巴结转移、远处转移的病例CD44v6表达率及表达强度高于未发生相应转移者，支持了CD44v6是转移相关因子的观点. 单因素生存分析结果显示，CD44v6阳性患者的五年生存率低于阴性患者,但在多因素生存分析中，CD44v6未进入最终模型，目前尚不能认为其是影响结直肠癌预后的独立因素. 而CD44v3的表达除与淋巴结转移、远处转移密切相关外，多因素生存分析显示CD44v3是影响结直肠癌预后的独立因素，提示与CD44v6相比，CD44v3不仅是转移相关因子，还是一个更有前途的结直肠癌预后标记物，但尚需进一步的前瞻性研究证实.
关于CD44v6参与肿瘤转移的机制，有学者提出假说认为为了与其他分子进行相互作用，转移的肿瘤细胞可能会以与激活的淋巴细胞相同或相似的机制利用CD44v6变异体结合到淋巴结的特异配体上，在淋巴结中得以停留，从而促进细胞生长及分化[45，46]. 李祖国等对CD44v6在大肠癌转移中的作用机制进行研究指出：CD44v6能促进大肠癌细胞与血管内皮细胞及基底膜的粘附和浸润，并影响细胞骨架蛋白的构型和分布，增强肿瘤细胞的运动能力，促进肿瘤转移的发生[47，48]. 而CD44v3则可能是由于其经硫酸肝素侧链修饰后，通过呈递生长因子(如肝素结合生长因子等)，增强生长因子与其受体的结合，从而促进细胞增殖和转移[49]. 目前对于不同CD44v参与不同肿瘤转移的确切机制仍不明确，有待于进一步研究探讨.
4 参考文献
1 Incidence of Malignant tumor in urban district of Shanghai in 1996. Department of Epidemiology of Shanghai Cancer
Institute. Zhongliu, 1999;19:inner back cover
2 Gu HP, Ni CR, Zhan RZ. Relationship of expressions of CD15, CD44v6 and nm23H1 mRNA with metastasis and prognosis of
colon carcinoma. Shijie Huaren Xiaohua Zazhi, 2000;8:887-891
3 Ratto C, Sofo L, Ippoliti M, Merico M, Doglietto GB, Crucitti F. Prognostic factors in colorectal cancer. Literature review for
clinical application. Dis Colon Rectum, 1998;41:1033-1049
4 McLeod HL, Murray GI. Tumour markers of prognosis in colorectal cancer. Br J Cancer, 1999;79:191-203
5 Goodison S, Tarin D. Current status of CD44 variant isoforms as cancer diagnostic markers. Histopathology, 1998;32:1-6
6 Tarin D, Matsumura Y. Deranged CD44 gene activity in malignancy. J Pathol, 1993;171:249-250
7 Matsumura Y, Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet,1992;340:1053-1058
8 Albelda SM. Biology of disease: role of integrins and other cell adhesion molecules in tumor progression and metastasis.
Lab Inves, 1993;68:4-17
9 East JA, Hart IR. Comments and critique: CD44 and its role in tumour progression and metastasis. Eur J Cancer,1993;29:1921-1922
10 Wielenga VJM, Heider KH, Offerhaus GJA, Adolf GR, van den Berg FM, Ponta H, Herrlich P, Pals ST. Expression of CD44 variant
proteins in human colorectal cancer is related to tumor progression. Cancer Res, 1993;53:4754-4756
11 Yamaguchi A, Urano T, Goi T, Saito M, Takeuchi K, Hirose K, Nakagawara G, Shiku H, Furukawa K. Expression of a CD44
variant containing exons 8 to 10 is a useful independent factor for the prediction of prognosis in colorectal cancer patients.
J Clin Oncol, 1996;14:1122-1127
12 Yamaguchi A, Goi T, Seki K, Ohtaki N, Maehara M, Kobayashi T, Niimoto S, Katayama K, Hirose K, Nakagawara G, Matsukawa S.
Clinical significance of combined immunohistochemical detection of CD44v and Sialyl Lex expression for colorectal cancer
patients undergoing curative resection. Oncology, 1998;55:400-404
13 Castell EM, Ariza A, Pellicer I, Fernndez-Vasalo A, Ojanguren I. Differential expression of CD44v6 in metastases of intestinal
and diffuse types of gastric carcinoma. J Clin Pathol, 1998;51:134-137
14 Yamamichi K, Uehara Y, Kitamura N, Nakane Y, Hioki K. Increased expression of CD44v6 mRNA significantly correlates with
distant metastasis and poor prognosis in gastric cancer. Int J Cancer, 1998;79:256-262
15 Washington K, Gottfried MR, Telen MJ. Expression of the cell adhesion molecule CD44 in gastric adenocarcinomas.
Hum Pathol, 1994;25:1043-1049
16 Mi JQ, Zhang ZH, Shen MC. Significance of CD44v6 protein expression in gastric carcinoma and precancerous lesions.
Shijie Huaren Xiaohua Zazhi, 2000;8:156-158
17 Xin Y, Zhao FK, Zhang SM, Wu DY, Wang YP, Xu L. Relationship between CD44v6 expression and prognosis in gastric
carcinoma patients. Shijie Huaren Xiaohua Zazhi, 1999;7:210-214
18 Chen GY, Wang DR. The expression and clinical significance of CD44v in human gastric cancers.
World J Gastroenterol, 2000;6:125-127
19 Kaufmann M, Heider KH, Sinn HP, Minckwitz GV, Ponta H, Herrlich P. CD44 variant exon epitopes in primary breast cancer and
length of survival. Lancet, 1995;345:615-619
20 Charpin C, Garcia S, Bouvier C, Devictor B, Andrac L, Choux R, Lavaut MN, Allasia C. Automated and quantitative
immunocytochemical assays of CD44v6 in breast carcinomas. Hum Pathol, 1997;28:289-296
21 Sinn HP, Heider KH, Skroch-Angel P, Minckwitz GV, Kaufmann M, Herrlich P, Ponta H. Human mammary carcinomas
express homologues of rat metastasis-associated variants of CD44. Breast Cancer Res Treat, 1995;36:307-313
22 Xiao CZ, Dai YM, Yu HY, Wang JJ, Ni CR. Relationship between expressions of CD44v6 and nm23-H1 and tumor invasion
and metastasis in hepatocellular carcinoma. Huaren Xiaohua Zazhi, 1998;6:1036-1038
23 Mathew J, Hines JE, Obafunwa JO, Burr AW, Toole K, Burt AD. CD44 is expressed in hepatocellular carcinomas showing
vascular invasion. J Pathol, 1996;179:74-79
24 Chen J, Zhang ZY, Zhu JQ, Wang CW, Xie Y, Huang DQ. Expression of CD44v6 in esophageal carcinoma and its clinical
significance. Huaren Xiaohua Zazhi, 1998;6:534
25 Borland G, Ross JA, Guy K. Forms and functions of CD44. Immunology, 1998;93:139-148
26 Ponta H, Wainwright D, Herrlich P. The CD44 protein family. Int J Biochem Cell Bio, 1998;30:299-305
27 Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI. Genomic structure of DNA encoding the lymphocyte homing
receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA, 1992;89:12160-12164
28 Tlg C, Hofmann M, Herrlich P, Ponta H. Splicing choice from ten variant exons establishes CD44 variability.
Nucl Acids Res, 1993;21:1225-1229
29 Günthert U, Hofmann M, Rudy W, Reber S, Zller M, Hauβmann I, Matzku S, Wenzel A, Ponta H, Herrlich P. A new variant
of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell, 1991;65:13-24
30 Rudy W, Hofmann M, Schwartz-Albiez R, Zller M, Heider KH, Ponta H, Herrlich P. The two major CD44 proteins expressed on
a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic
behavior. Cancer Res, 1993;53:1262-1268
31 Seiter S, Arch R, Reber S, Komitowski D, Hofmann M, Ponta H, Herrlich P, Matzku S, Zller M. Prevention of tumor
metastasis formation by anti-variant CD44. J Exp Med, 1993;177:443-455
32 Herold-Mende C, Seiter S, Born AI, Patzelt E, Schupp M, Zller J, Bosch FX, Zller M. Expression of CD44 splice variants in
squamous epithelia and squamous cell carcinomas of the head and neck. J Pathol, 1996;179:66-73
33 Saegusa M, Hashimura M, Machida D, Okayasu I. Down-regulation of CD44 standard and variant isoforms during the development
and progression of uterine cervical tumours. J Pathol, 1999;187:173-183
34 Speiser P, Wanner C, Tempeer C, Mittelbck M, Hanzal E, Bancher-Todesca D, Gitsch G, Reinthaller A, Kainz C. CD44 is
an independent prognostic factor in early-stage cervical cancer. Int J Cancer, 1997;74:185-188
35 Mulder JWR, Kruyt PM, Sewnath M, Oosting J, Seldenrijk CA, Weidema WF, Offerhaus GJA, Pals ST. Colorectal cancer prognosis
and expression of exon-v6-containing CD44 proteins. Lancet, 1994;344:1470-1472
36 Wielenga VJM, van Der Voort R, Mulder JWR, Kruyt PM, Weidema WF, Oosting J, Seldenrijk CA, Krimpen CV, Offerhaus GJA.
CD44 splice variants as prognostic markers in colorectal cancer. Scand J Gastroenterol, 1998;33:82-87
37 Gotley DC, Fawcett J, Walsh MD, Reeder JA, Simmons DL, Antalis TM. Alternatively spliced variants of the cell adhesion molecule
CD44 and tumor progression in colorectal cancer. Br J Cancer, 1996;74:342-351
38 Givehchian M, Wrner S, Strter J, Zller M, Heuschen U, Heuschen G, Lehnert T, Herfarth C, von knebler Doeberitz M.
No evidence for cancer-related CD44 splice variants in primary and metastatic colorectal cancer. Eur J Cancer,1998;34:1099-1104
39 Finke LH, Terpe HJ, Zrb C, Haensch W, Schlag PM. Colorectal cancer prognosis and expression of exon-v6-containing CD44
proteins. Lancet, 1995;345:583
40 Koretz K, Moller P, Lehnert T, Hinz U, Otto HHF, Herfarth C. Effect of CD44v6 on survival in colorectal carcinoma.
Lancet, 1995;345:327-328
41 Coppola D, Hyacinthe M, Fu L, Cantor AB, Karl R, Marcet J, Cooper DL, Nicosia SV, Cooper HS. CD44v6 expression in
human colorected carcinoma. Hum Pathol, 1998;29:627-635
42 Weg-Remers S, Anders M, von Lampe B, Riecken EO, Schüder G, Feifel G, Zeitz M, Stallmach A. Decreased expression of CD44
splicing variants in advanced colorectal carcinomas. Eur J Cancer, 1998;34:1607-1611
43 Ropponen KM, Eskelinen MJ, Lipponen PK, Alhava E, Kosma VM. Expression of CD44 and variant proteins in human colorectal and
its relevance for prognosis. Scand J Gastroenterol, 1998;33:301-309
44 Kalish ED, Iida N, Moffat FL, Bourguignon LY. A new CD44V3-containing isoform is involved in tumor cell growth and migration
during human breast carcinoma progression. Front Biosci, 1999;4:A1-8
45 Herrlich P, Zller M, Pals ST, Ponta H. CD44 splice variants: metastases meet lymphocytes. Immunol Today,
1993;14:395-399
46 Arch R, Wirth K, Hofmann M, Ponta H, Matzku S, Herrlich P, Zller M. Participation in normal immune responses of
a metastasis-inducing splice variant of CD44. Science, 1992;257:682-685
47 Li ZG, Ding YQ, Deng YJ, Guo JQ, Zhu MG. Mechanism of CD44V6 in human colorectal carcinoma metastasis.
Zhonghua Yixue Zazhi, 1998；78:729-732
48 Li ZG, Ding YQ, Deng YJ, Zhu MG. Expression of CD44V6 and its effects on cytoskeleton in human colorectal carcinoma
cell lines. Zhonghua Binglixue Zazhi, 1998;27:273-277
49 Bennett KL, Jackson DG, Simon JC, Tanczos E, Peach R, Modrell B, Stamenkovic I, Plowman G, Aruffo A. CD44 isoforms
containing exon V3 are responsible for the presentation of heparin-binding growth factor. J Cell Bio, 1995;128:687-698, http://www.100md.com(蔡 崎 陆洪芬 孙孟红 杜 祥 范月珍 施达仁)
蔡崎，女，1972-01-16生，辽宁省大连市人，汉族. 1994年上海医科大学医学系本科毕业. 现复旦大学医学院(原上海医科大学)附属肿瘤医院肿瘤病理专业博士在读. 研究方向：胃肠道肿瘤的病理学和肿瘤转移.
项目负责人 蔡崎，200032，上海市零陵路399号，复旦大学医学院附属肿瘤医院病理科.
Department of Pathology, Cancer Hospital/Cancer Institute of Medical Center of Fudan University, Shanghai 200032, China
Correspondence to Qi Cai, Department of Pathology, Cancer Hospital/Cancer Institute of Medical Center of Fudan University,Shanghai 200032, China
Tel. 0086-21-64175590 Ext.3357
Email. caiqi@baoscape.com
Received 2000-05-18 Accepted 2000-06-02
Expression of CD44 v3 and v6 proteins in human colorectal carcinoma and its relevance with prognosis
Qi Cai, Hong-Fen Lu, Meng-Hong Sun, Xiang Du, Yue-Zhen Fan and Da-Ren Shi
Abstract
AIM To evaluate the expression of CD44v3 and v6 protein in colorectal carcinoma and its prognostic significance.
METHODS One hundred and twenty-one cases of formalin-fixed paraffin-embedded colorectal carcinoma were retrospectively analyzed using EnvisionTM immunohistochemical method with the monoclonal antibody CD44v3 and v6. The median following-up time was 67.77 months and the prognostic value of the CD44v3 and CD44v6 was assessed using univariate and multivariate survival analysis.
RESULTS The positive rates of CD44v3 and v6 protein were 60.3% and 57.9%, respectively. There was significant correlation between CD44v3 immunoreactivity and tumor location,lymph node metastasis, distant metastasis and Duke＇s stage (P<0.05, Spearman correlation test). Significant correlation between CD44v6 immunoreactivity and patients＇ gender, lymph node metastasis, distant metastasis, Duke＇s stage was also noticed (P<0.05, Spearman correlation test). The 5-year survival rates were 81.25% and 60.27% in CD44v3 negative cases and CD44v3 positive ones, respectively. As CD44v6, the 5-year survival rates were 80.39% and 60.00% in CD44v6 negative cases and CD44v6 positive ones, respectively; the differences between the two groups of patients were significant (P<0.05, Log-rank test). In multivariate analysis using the Cox regression model, CD44v3 expression emerges as an independent prognostic indicator.
CONCLUSION CD44v3 and v6 might play some important roles in metastasis of colorectal carcinoma, and CD44v3 expression might be a new useful independent prognostic marker of colorectal carcinoma.
Subject headings colorectal neoplasms; cell adhesion molecules; CD44v3; CD44v6; neoplasm metastasis; immunohistochemistry
Cai Q, Lu HF, Sun MH, Du X, Fan YZ, Shi DR. Expression of CD44 v3 and v6 proteins in human colorectal carcinoma and its relevance with prognosis. Shijie Huaren Xiaohua Zazhi, 2000;8(11):1255-1258
摘要
目的 研究CD44v3, v6蛋白在结直肠癌组织中的表达及其预后意义.
方法 应用EnvisionTM 免疫组化法，回顾性分析121例结直肠癌石蜡组织CD44v3, v6的表达. 患者中位随访时间为67.77mo.
结果 CD44v3, v6在结直肠癌中的阳性率分别为60.3%和57.9%.CD44v3的阳性表达与患者肿瘤部位，淋巴结转移状况、远处转移与否、临床分期密切相关(P<0.05，Spearman等级相关检验). CD44v6的阳性表达与患者性别、淋巴结转移状况、远处转移与否、临床分期密切相关(P<0.05，Spearman等级相关检验). Kaplan-Meier生存曲线，Log-rank检验单因素生存分析结果显示，CD44v3阴性、阳性患者的五年生存率分别为81.25%, 60.27%，两者之间存在显著性差异(P=0.035)；CD44v6阴性、阳性患者的5a生存率分别为80.39%, 60.00%，两者之间亦存在显著性差异(P=0.0299). Cox模型多因素生存分析结果显示，CD44v3表达是影响结直肠癌预后的独立因素.
结论 CD44v3, v6蛋白与结直肠癌转移、预后相关，CD44v3是影响结直肠癌预后的独立因素.
主题词 结肠直肠肿瘤；细胞粘附分子；CD44v3；CD44v6；肿瘤转移；免疫组织化学
蔡崎, 陆洪芬, 孙孟红, 杜祥, 范月珍, 施达仁. 结直肠癌组织中CD44v3, v6蛋白的表达意义. 世界华人消化杂志，2000；8(11)：1255-1258
0 引言
结直肠癌是一种常见的消化道恶性肿瘤，近年来发病率不断增加. 以上海为例，上海市肿瘤研究所流行病学研究室统计资料显示：1996年结直肠癌发病率为69.9/10万，并有继续上升趋势[1]. 转移的发生是导致结直肠癌患者死亡的主要原因，是临床治疗的最大难题之一，影响着患者的预后. 对结直肠癌转移潜能、预后的有效评估非常重要，能够指导临床治疗方案的制定，不仅可使适宜的患者及时接受必要的辅助治疗，也可使某些患者避免承受不必要的放疗、化疗所致的副作用，从而提高患者的生存率和生存质量[2-4]. 近年来细胞粘附分子CD44,尤其CD44变异体(CD44v)与肿瘤转移的关系正日益受到人们的重视[5-24]. 然而结直肠癌CD44v方面研究报道尚少，且结果不一. 故本文采用免疫组化方法检测结直肠癌组织中CD44v3,v6蛋白的表达情况，并观察其与转移、预后的关系.
1 材料和方法
1.1 材料 我院1991-01/1994-04临床病理资料完整的结直肠癌121例(结肠癌50例，直肠癌71例). 其中男72例，女49例. 年龄22～88(平均55，中位57)岁. 高分化腺癌8例，中分化腺癌89例，低分化腺癌9例，粘液腺癌14例，印戒细胞癌1例. Duke＇s A期20例，Duke＇s B期31例，Duke＇s C 期58例，Duke＇s D期12例. 随访至1999-04，死亡42例，存活79例，存活时间1.50～100.27(平均59.75，中位67.77)mo. 所有病例术前均未经放疗和化疗，并由两位病理医生确诊. 另取40例距癌10cm以远处肠粘膜作对照研究. 抗CD44v3,抗CD44v6mAb购自Bender Medsystem 公司，工作稀释度分别为1∶40和1∶400. Envision反应液购自Dako公司.
1.2 方法 取存档蜡块制备4μm连续切片，二甲苯脱蜡、梯度酒精水化后置于pH=6.0的枸橼酸盐缓冲液中，微波法修复抗原：95℃, 5min×4次，EnvisionTM免疫组化法染色. DAB显色，苏木素复染，常规脱水、透明、封片，观察结果. 用TBS代替一抗作阴性对照，正常皮肤鳞状上皮作阳性对照. CD44v3阳性定位于胞质，CD44v6阳性定位于胞膜. 免疫组化结果采用半定量计分法判定. 即A.按阳性着色程度评分. 0分：无着色；1分：浅黄色；2分：棕黄色；3分：棕褐色. B.按阳性细胞所占比例评分. 0分：<5%；1分：5%～10%; 2分：11%～50%；3分：51%～80%；4分：>80%. 两者乘积：0分为阴性(-)；1～4分为弱阳性(+)；5～8分为中度阳性(++)；9～12分为强阳性(+++).
统计学处理 应用SPSS8.2软件spearman相关分析. 生存分析采用Kaplan-meier生存曲线、Log-rank单因素分析及Cox风险模型多因素分析.
2 结果
正常肠粘膜均不表达CD44v3, v6. 结直肠癌组织CD44v3表达阳性率为60.4%. 其中+者占43.0%，++者占14.9%，+++者占2.5%. CD44v6表达阳性率为57.9%. 其中+者占43.8%，++者占10.7%，+++者占3.3%. 且CD44v3与CD44v6的表达密切相关
(P<0.05，表1).
表1 CD44v6, v3表达的相关性
| CD44v3 | n | |||
| - | + | ++～+++ | ||
| CD44v6 (-) | 29 | 20 | 2 | 51 |
| CD44v6 (+) | 17 | 26 | 10 | 53 |
| CD44v6 (++～+++) | 2 | 6 | 9 | 17 |
| n | 48 | 52 | 21 | 121 |
P=0.000, r=0.411, Spearman 相关分析.
2.1 CD44v3, v6表达与临床病理特征 免疫组化结果显示，CD44v3阳性表达与患者年龄、性别、大体类型、组织学类型、肿块最大径、脉管神经侵犯、肿瘤浸润深度均无显著相关(P>0.05)，而与肿瘤部位、淋巴结转移状况、有无远处转移及Duke＇s分期密切相关(P<0.05，表2). CD44v6阳性表达与患者年龄、部位、大体类型、组织学类型、肿块最大径、脉管神经侵犯、肿瘤浸润深度均无显著相关(P>0.05)，而与患者性别、淋巴结转移状况、有无远处转移及Duke＇s分期密切相关(P<0.05，表2).
表2 CD44v6, v3的表达与结直肠癌患者临床病理特征的关系
| 临床病理 n CD44v6表达 CD44v3表达 |
| - + ++～+++ P - + ++～+++ P |
| 淋巴结 无转移 57 31 21 5 28 22 7 有转移 64 20 32 12 0.007* 20 30 14 0.034 * 远处转移 无 109 50 46 13 47 46 16 有 12 1 7 4 0.005* 1 6 5 0.005* Duke＇s分期 A 20 13 6 1 12 7 1 B 31 15 13 3 13 13 5 C 58 22 27 9 22 26 10 D 12 1 7 4 0.001* 1 6 5 0.004* 年龄 ≤40 14 4 8 2 5 5 4 >40 107 47 45 15 0.378 43 47 17 0.458 性别 男 72 39 26 7 31 30 11 女 49 12 27 10 0.001* 17 22 10 0.315 部位 直肠 71 29 31 11 34 30 7 结肠 50 22 22 6 0.631 14 22 14 0.005* 大体类型 非外生性 88 35 40 13 37 34 17 外生性 22 16 12 4 0.360 11 17 4 0.841 组织学类型 高及中分化 97 40 45 12 38 44 15 低分化及其他 24 11 8 5 0.938 10 8 6 0.764 肿块最大径 ≤5cm 82 33 38 11 37 30 15 >5cm 38 18 14 6 0.650 11 21 6 0.262 浸润深度 肌层及肌层内 28 14 10 4 33 40 18 肌层外 91 36 42 13 0.438 14 11 3 0.148 脉管侵犯 有 28 12 12 4 7 14 7 无 93 39 41 13 0.957 41 38 14 0.059 神经侵犯 有 20 10 8 2 7 12 1 无 101 41 45 15 0.404 41 40 20 0.744 |
Spearman 相关分析.
2.2 CD44v3, v6表达与生存率 绘制Kaplan-Meier生存曲线，采用Log-rank单因素检验对121例结直肠癌患者的生存情况进行分析，结果显示：CD44v3阴性患者的五年生存率为81.25%，阳性患者的五年生存率为60.27%，二者之间存在显著性差异(P=0.0035，图1). CD44v6阴性、阳性患者的五年生存率分别为80.39%, 60.00%，二者之间亦存在显著性差异(P=0.0299，图2). 此外，在临床病理特征中，肿瘤浸润深度，淋巴结转移状况，有无远处转移，神经侵犯及Duke＇s分期与患者生存情况相关(P<0.05). 而年龄，性别，大体类型，组织学类型，部位，脉管侵犯，肿块最大径与预后无显著性相关(P>0.05). 应用Cox模型进行多因素分析显示：Duke＇s分期，神经侵犯，CD44v3是影响结直肠癌预后的独立因素(表3)，而CD44v6未进入最终模型.
表3 Cox回归多因素生存分析
| 临床病理因素 | B S.E. Wald P R 95%CI |
| Duke＇s分期 神经侵犯情况 CD44v3表达 大体类型 肿块部位 CD44v6表达 肿块最大径 脉管侵犯情况 组织学类型 年龄 性别 | 1.4006 0.2972 22.2107 0.000* 0.2316 2.2662-7.2648 -1.2186 0.4113 8.7790 0.0030* -0.1341 0.1320-0.6620 0.8742 0.4143 4.4527 0.0348* 0.0807 1.0642-5.3985 -0.6682 0.4490 2.2141 0.1368 -0.0238 0.2126-1.2361 -0.4492 0.3783 1.4096 0.2351 0.0000 0.3040-1.3396 0.3495 0.3975 0.7731 0.3793 0.0000 0.6508-3.0915 -0.1968 0.2401 0.6714 0.4126 0.0000 0.5130-1.3151 -0.2702 0.3681 0.5389 0.4629 0.0000 0.3710-1.5702 0.3062 0.4476 0.4679 0.4940 0.0000 0.5649-3.2656 -0.1747 0.2810 0.3865 0.5341 0.0000 0.4842-1.4564 0.1476 0.4242 0.1210 0.7280 0.0000 0.5046-2.6618 |
图1 CD44v6表达与患者生存的关系(Log-rank test, P=0.0299).
图2 CD44v3表达与患者生存的关系(Log-rank test, P=0.0035).
3 讨论
恶性肿瘤细胞重要的生物学特征之一表现为细胞与细胞之间、细胞与细胞外基质之间粘附特性的异常，这种粘附特性的异常导致肿瘤细胞具有了从原发部位脱落并向周围组织侵袭及向远隔器官转移的能力. CD44正是一个近年来倍受关注，在细胞恶性转化过程中其结构和功能均发生显著变化而影响转化后肿瘤细胞侵袭转移行为的细胞表面粘附分子[17]. CD44作为具有高度异质性的单链跨膜糖蛋白，在体内分布极广泛，能与透明质酸等多种配体结合，参与细胞与细胞、细胞与基质之间的粘连. 其具有高度异质性，系编码该蛋白的基因在转录过程中的mRNA选择性拼接和蛋白质合成过程中的翻译后修饰不同所致. 主要有两种类型，标准型(CD44s)和变异型(CD44v)，后者又由于选择性拼接方式不同而包含有多种类型[25-28]. 近年来研究表明,CD44v可在不同水平被调节，参与肿瘤的侵袭和转移[6,25,26].
CD44v6作为较为公认的转移相关因子，最早由动物实验发现，将大鼠转移性胰腺癌细胞株(BSP73ASML)的CD44vDNA转染到非转移的BSP73AS癌细胞株中，发现后者发生了转移[29]，经测定CD44v4-7和CD44v6-7是BSP73ASML株中表达最强的两种CD44v[30]，而预先用抗CD44v抗体处理可能防止转移的发生[31]. 随后，不同学者用不同方法对不同肿瘤标本的不同CD44表达情况作了研究，发现大多数肿瘤如肠癌、胃癌、乳腺癌、肝癌等的CD44尤其CD44v表达增高，并在不同程度上与肿瘤的侵袭、转移相关[10-23]. 也有少数肿瘤如食管、头颈部及宫颈鳞癌则出现CD44v的负调节[24，32-34].
关于CD44v6与结直肠癌转移、预后关系的研究，不同学者结论不一. Wielenga et al[10]报道，含有v6序列的CD44表达通常限于肿瘤发展的后期，在转移性(Duke＇s C/D期)中的表达高于未发生转移者(Duke＇s A/B期). Mulder et al[35]研究亦显示CD44v6在肿瘤进展期有过度表达，CD44v6可作为一种独立的估计结直肠癌预后的标记物，并可用以指导Duke＇s B, C期结直肠癌治疗方案的制定. Wielenga et al[36]用免疫组化法(IHC)证实CD44v6在结直肠癌中的表达具有显著的预后意义. 而Gotley et al[37], Givehchian et al[38]研究显示CD44v与结直肠癌恶性进展和转移无关. Finke et al[39], Koretz et al[40], Coppola et al[41]认为CD44v6不能作为结直肠癌的预后指标. Weg-Remers et al[42]甚至研究显示CD44v6在转移性肠癌和转移灶中表达降低. 笔者对上述研究进行分析后认为，不同学者得出不同结论的原因可能如下：①肿瘤本身的异质性；取材的偏差. ②各实验室的操作不同，同时所用的试剂不同，各实验室所订的标准不一样. ③IHC方法所用的抗体特异性高低不一. ④研究方法不同，灵敏度存在差异. ⑤首先报道者可能会对所取得的结论过分估计其真正价值等等.
对CD44v3与肿瘤转移、预后关系的研究较少，Ropponen et al[43]认为其可能作为结直肠癌的预后指标. 而在对与乳腺癌进展、转移关系的研究中，Kalish et al[44]指出含有v3变异体的CD44v与乳腺癌的转移密切相关.
我们采用EnvisionTM免疫组化两步法，敏感度、特异性较高，背景清晰. 结果显示：与正常肠粘膜相比，CD44v6在结直肠癌中的表达显著升高. CD44v6的表达与患者年龄、性别、肿瘤大小等均无相关，而与淋巴结转移、远处转移密切相关，发生淋巴结转移、远处转移的病例CD44v6表达率及表达强度高于未发生相应转移者，支持了CD44v6是转移相关因子的观点. 单因素生存分析结果显示，CD44v6阳性患者的五年生存率低于阴性患者,但在多因素生存分析中，CD44v6未进入最终模型，目前尚不能认为其是影响结直肠癌预后的独立因素. 而CD44v3的表达除与淋巴结转移、远处转移密切相关外，多因素生存分析显示CD44v3是影响结直肠癌预后的独立因素，提示与CD44v6相比，CD44v3不仅是转移相关因子，还是一个更有前途的结直肠癌预后标记物，但尚需进一步的前瞻性研究证实.
关于CD44v6参与肿瘤转移的机制，有学者提出假说认为为了与其他分子进行相互作用，转移的肿瘤细胞可能会以与激活的淋巴细胞相同或相似的机制利用CD44v6变异体结合到淋巴结的特异配体上，在淋巴结中得以停留，从而促进细胞生长及分化[45，46]. 李祖国等对CD44v6在大肠癌转移中的作用机制进行研究指出：CD44v6能促进大肠癌细胞与血管内皮细胞及基底膜的粘附和浸润，并影响细胞骨架蛋白的构型和分布，增强肿瘤细胞的运动能力，促进肿瘤转移的发生[47，48]. 而CD44v3则可能是由于其经硫酸肝素侧链修饰后，通过呈递生长因子(如肝素结合生长因子等)，增强生长因子与其受体的结合，从而促进细胞增殖和转移[49]. 目前对于不同CD44v参与不同肿瘤转移的确切机制仍不明确，有待于进一步研究探讨.
4 参考文献
1 Incidence of Malignant tumor in urban district of Shanghai in 1996. Department of Epidemiology of Shanghai Cancer
Institute. Zhongliu, 1999;19:inner back cover
2 Gu HP, Ni CR, Zhan RZ. Relationship of expressions of CD15, CD44v6 and nm23H1 mRNA with metastasis and prognosis of
colon carcinoma. Shijie Huaren Xiaohua Zazhi, 2000;8:887-891
3 Ratto C, Sofo L, Ippoliti M, Merico M, Doglietto GB, Crucitti F. Prognostic factors in colorectal cancer. Literature review for
clinical application. Dis Colon Rectum, 1998;41:1033-1049
4 McLeod HL, Murray GI. Tumour markers of prognosis in colorectal cancer. Br J Cancer, 1999;79:191-203
5 Goodison S, Tarin D. Current status of CD44 variant isoforms as cancer diagnostic markers. Histopathology, 1998;32:1-6
6 Tarin D, Matsumura Y. Deranged CD44 gene activity in malignancy. J Pathol, 1993;171:249-250
7 Matsumura Y, Tarin D. Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet,1992;340:1053-1058
8 Albelda SM. Biology of disease: role of integrins and other cell adhesion molecules in tumor progression and metastasis.
Lab Inves, 1993;68:4-17
9 East JA, Hart IR. Comments and critique: CD44 and its role in tumour progression and metastasis. Eur J Cancer,1993;29:1921-1922
10 Wielenga VJM, Heider KH, Offerhaus GJA, Adolf GR, van den Berg FM, Ponta H, Herrlich P, Pals ST. Expression of CD44 variant
proteins in human colorectal cancer is related to tumor progression. Cancer Res, 1993;53:4754-4756
11 Yamaguchi A, Urano T, Goi T, Saito M, Takeuchi K, Hirose K, Nakagawara G, Shiku H, Furukawa K. Expression of a CD44
variant containing exons 8 to 10 is a useful independent factor for the prediction of prognosis in colorectal cancer patients.
J Clin Oncol, 1996;14:1122-1127
12 Yamaguchi A, Goi T, Seki K, Ohtaki N, Maehara M, Kobayashi T, Niimoto S, Katayama K, Hirose K, Nakagawara G, Matsukawa S.
Clinical significance of combined immunohistochemical detection of CD44v and Sialyl Lex expression for colorectal cancer
patients undergoing curative resection. Oncology, 1998;55:400-404
13 Castell EM, Ariza A, Pellicer I, Fernndez-Vasalo A, Ojanguren I. Differential expression of CD44v6 in metastases of intestinal
and diffuse types of gastric carcinoma. J Clin Pathol, 1998;51:134-137
14 Yamamichi K, Uehara Y, Kitamura N, Nakane Y, Hioki K. Increased expression of CD44v6 mRNA significantly correlates with
distant metastasis and poor prognosis in gastric cancer. Int J Cancer, 1998;79:256-262
15 Washington K, Gottfried MR, Telen MJ. Expression of the cell adhesion molecule CD44 in gastric adenocarcinomas.
Hum Pathol, 1994;25:1043-1049
16 Mi JQ, Zhang ZH, Shen MC. Significance of CD44v6 protein expression in gastric carcinoma and precancerous lesions.
Shijie Huaren Xiaohua Zazhi, 2000;8:156-158
17 Xin Y, Zhao FK, Zhang SM, Wu DY, Wang YP, Xu L. Relationship between CD44v6 expression and prognosis in gastric
carcinoma patients. Shijie Huaren Xiaohua Zazhi, 1999;7:210-214
18 Chen GY, Wang DR. The expression and clinical significance of CD44v in human gastric cancers.
World J Gastroenterol, 2000;6:125-127
19 Kaufmann M, Heider KH, Sinn HP, Minckwitz GV, Ponta H, Herrlich P. CD44 variant exon epitopes in primary breast cancer and
length of survival. Lancet, 1995;345:615-619
20 Charpin C, Garcia S, Bouvier C, Devictor B, Andrac L, Choux R, Lavaut MN, Allasia C. Automated and quantitative
immunocytochemical assays of CD44v6 in breast carcinomas. Hum Pathol, 1997;28:289-296
21 Sinn HP, Heider KH, Skroch-Angel P, Minckwitz GV, Kaufmann M, Herrlich P, Ponta H. Human mammary carcinomas
express homologues of rat metastasis-associated variants of CD44. Breast Cancer Res Treat, 1995;36:307-313
22 Xiao CZ, Dai YM, Yu HY, Wang JJ, Ni CR. Relationship between expressions of CD44v6 and nm23-H1 and tumor invasion
and metastasis in hepatocellular carcinoma. Huaren Xiaohua Zazhi, 1998;6:1036-1038
23 Mathew J, Hines JE, Obafunwa JO, Burr AW, Toole K, Burt AD. CD44 is expressed in hepatocellular carcinomas showing
vascular invasion. J Pathol, 1996;179:74-79
24 Chen J, Zhang ZY, Zhu JQ, Wang CW, Xie Y, Huang DQ. Expression of CD44v6 in esophageal carcinoma and its clinical
significance. Huaren Xiaohua Zazhi, 1998;6:534
25 Borland G, Ross JA, Guy K. Forms and functions of CD44. Immunology, 1998;93:139-148
26 Ponta H, Wainwright D, Herrlich P. The CD44 protein family. Int J Biochem Cell Bio, 1998;30:299-305
27 Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell JI. Genomic structure of DNA encoding the lymphocyte homing
receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA, 1992;89:12160-12164
28 Tlg C, Hofmann M, Herrlich P, Ponta H. Splicing choice from ten variant exons establishes CD44 variability.
Nucl Acids Res, 1993;21:1225-1229
29 Günthert U, Hofmann M, Rudy W, Reber S, Zller M, Hauβmann I, Matzku S, Wenzel A, Ponta H, Herrlich P. A new variant
of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell, 1991;65:13-24
30 Rudy W, Hofmann M, Schwartz-Albiez R, Zller M, Heider KH, Ponta H, Herrlich P. The two major CD44 proteins expressed on
a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic
behavior. Cancer Res, 1993;53:1262-1268
31 Seiter S, Arch R, Reber S, Komitowski D, Hofmann M, Ponta H, Herrlich P, Matzku S, Zller M. Prevention of tumor
metastasis formation by anti-variant CD44. J Exp Med, 1993;177:443-455
32 Herold-Mende C, Seiter S, Born AI, Patzelt E, Schupp M, Zller J, Bosch FX, Zller M. Expression of CD44 splice variants in
squamous epithelia and squamous cell carcinomas of the head and neck. J Pathol, 1996;179:66-73
33 Saegusa M, Hashimura M, Machida D, Okayasu I. Down-regulation of CD44 standard and variant isoforms during the development
and progression of uterine cervical tumours. J Pathol, 1999;187:173-183
34 Speiser P, Wanner C, Tempeer C, Mittelbck M, Hanzal E, Bancher-Todesca D, Gitsch G, Reinthaller A, Kainz C. CD44 is
an independent prognostic factor in early-stage cervical cancer. Int J Cancer, 1997;74:185-188
35 Mulder JWR, Kruyt PM, Sewnath M, Oosting J, Seldenrijk CA, Weidema WF, Offerhaus GJA, Pals ST. Colorectal cancer prognosis
and expression of exon-v6-containing CD44 proteins. Lancet, 1994;344:1470-1472
36 Wielenga VJM, van Der Voort R, Mulder JWR, Kruyt PM, Weidema WF, Oosting J, Seldenrijk CA, Krimpen CV, Offerhaus GJA.
CD44 splice variants as prognostic markers in colorectal cancer. Scand J Gastroenterol, 1998;33:82-87
37 Gotley DC, Fawcett J, Walsh MD, Reeder JA, Simmons DL, Antalis TM. Alternatively spliced variants of the cell adhesion molecule
CD44 and tumor progression in colorectal cancer. Br J Cancer, 1996;74:342-351
38 Givehchian M, Wrner S, Strter J, Zller M, Heuschen U, Heuschen G, Lehnert T, Herfarth C, von knebler Doeberitz M.
No evidence for cancer-related CD44 splice variants in primary and metastatic colorectal cancer. Eur J Cancer,1998;34:1099-1104
39 Finke LH, Terpe HJ, Zrb C, Haensch W, Schlag PM. Colorectal cancer prognosis and expression of exon-v6-containing CD44
proteins. Lancet, 1995;345:583
40 Koretz K, Moller P, Lehnert T, Hinz U, Otto HHF, Herfarth C. Effect of CD44v6 on survival in colorectal carcinoma.
Lancet, 1995;345:327-328
41 Coppola D, Hyacinthe M, Fu L, Cantor AB, Karl R, Marcet J, Cooper DL, Nicosia SV, Cooper HS. CD44v6 expression in
human colorected carcinoma. Hum Pathol, 1998;29:627-635
42 Weg-Remers S, Anders M, von Lampe B, Riecken EO, Schüder G, Feifel G, Zeitz M, Stallmach A. Decreased expression of CD44
splicing variants in advanced colorectal carcinomas. Eur J Cancer, 1998;34:1607-1611
43 Ropponen KM, Eskelinen MJ, Lipponen PK, Alhava E, Kosma VM. Expression of CD44 and variant proteins in human colorectal and
its relevance for prognosis. Scand J Gastroenterol, 1998;33:301-309
44 Kalish ED, Iida N, Moffat FL, Bourguignon LY. A new CD44V3-containing isoform is involved in tumor cell growth and migration
during human breast carcinoma progression. Front Biosci, 1999;4:A1-8
45 Herrlich P, Zller M, Pals ST, Ponta H. CD44 splice variants: metastases meet lymphocytes. Immunol Today,
1993;14:395-399
46 Arch R, Wirth K, Hofmann M, Ponta H, Matzku S, Herrlich P, Zller M. Participation in normal immune responses of
a metastasis-inducing splice variant of CD44. Science, 1992;257:682-685
47 Li ZG, Ding YQ, Deng YJ, Guo JQ, Zhu MG. Mechanism of CD44V6 in human colorectal carcinoma metastasis.
Zhonghua Yixue Zazhi, 1998；78:729-732
48 Li ZG, Ding YQ, Deng YJ, Zhu MG. Expression of CD44V6 and its effects on cytoskeleton in human colorectal carcinoma
cell lines. Zhonghua Binglixue Zazhi, 1998;27:273-277
49 Bennett KL, Jackson DG, Simon JC, Tanczos E, Peach R, Modrell B, Stamenkovic I, Plowman G, Aruffo A. CD44 isoforms
containing exon V3 are responsible for the presentation of heparin-binding growth factor. J Cell Bio, 1995;128:687-698, http://www.100md.com(蔡 崎 陆洪芬 孙孟红 杜 祥 范月珍 施达仁)