【作者】 白艳花；

【导师】 周庚寅； 觉道建一；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 背景与目的甲状腺乳头状癌是人体最常见的内分泌恶性肿瘤。甲状腺癌患者的预后较好,术后10年无病生存率约为90%,但仍有10%的患者出现术后癌复发、远处转移甚至因癌死亡。研究表明,手术时甲状腺外浸润的出现与患者术后的不良预后密切相关。因此,发现影响甲状腺乳头状癌发展和浸润的分子水平的变化,对甲状腺乳头状癌的治疗具有重要应用意义。最近,一个新基因periostin,又称osteoblast specific protein 2(OSF-2)被连续报道在一系列癌的生长,血管生成,浸润及转移中起重要角色,包括甲状腺乳头状癌。在乳腺癌培养细胞及小鼠种植瘤实验中,periostin的高表达可导致血管内皮生长因子2的高表达,从而推动癌的进展和血管生成;在肺非小细胞癌中,患者血清中periostin的高水平与不良临床预后之间有显著统计学意义;在甲状腺乳头状癌组织中,periostin的高表达与癌的甲状腺外浸润、淋巴结转移和癌的高进展阶段相关。进一步研究发现,将periostin基因导入到293T癌细胞后,癌细胞会出现上皮细胞间质转化,从而侵袭活性增强。有报道表明,在鳞状细胞癌培养细胞中,上皮细胞间质转化表型的癌细胞,其periostin的表达量远高于上皮表型的癌细胞。上皮细胞间质转化是癌浸润转移过程中的一个普遍现象,其特征为浸润前沿部癌细胞上皮特性的丧失和间质特性的获得,包括形态学方面癌细胞极性缺失和/或粘附性下降,及分子水平E-cadherin表达下降,vimentin、上皮生长因子受体、间质金属蛋白酶-9、TGF-β、NF_kβ表达上调等。然而,在甲状腺乳头状癌中,作为上皮细胞间质转化形态学特征的癌细胞极性缺失和/或粘附性下降与其他临床病理指标的关系,包括与甲状腺外浸润及淋巴结转移的关系仍然未知;与periostin表达的关系也依然未知。到目前为止,人体组织中periostin共有五种isoform被报道。这五种isoform的差异位于外显子ⅩⅥ～ⅩⅫ(对应于periostin蛋白的C端),由转录水平mRNA的选择性剪接(alternative splicing)造成。在膀胱癌组织和正常组织中,有不同的periostin isoform表达,因此periostin isoform的表达方式被认为与膀胱癌的发生有关。periostin isoform在甲状腺组织,包括癌组织和非癌组织中的表达尚未有道。本研究拟解决以下几个问题:(1)甲状腺乳头状癌浸润前沿癌细胞极性缺失和/或粘附性下降与其它临床病理学指标(性别、年龄、癌的大小、甲状腺外浸润、淋巴结转移等)的关系及其对临床预后的影响。(2)甲状腺乳头状癌浸润前沿癌细胞极性缺失和/或粘附性下降与periostin表达的关系。(3) periostin isoform在甲状腺癌组织及非癌组织中的表达方式及其与甲状腺癌的发生或发展的关系。方法1.将68例甲状腺乳头状癌癌组织及其周围组织用甲醛固定,石蜡包埋,制成HE切片,检测浸润前沿部癌细胞极性缺失和/或粘附性下降的情况,并用X~2检验或Fisher’s精确概率检验检测其与其它临床病理学指标(性别、年龄、癌的大小、甲状腺外浸润、淋巴结转移等)的关系。2.提取1中所述病例的甲状腺癌组织及其背景甲状腺非癌组织的全RNA,逆转录成cDNA,用RT-PCR法检测是否有periostin表达。用Taqman的探针和引物,采用real-time PCR法检测periostin在甲状腺组织的定量表达。β-actin为内对照。periostin在甲状腺乳头状癌癌组织和对应的非癌组织中的差异用ΔΔCt法计算,表示为T/N比(T:癌组织;N:非癌组织)。用χ~2检验或Fisher’s精确概率检验检测periostin的表达与浸润前沿部癌细胞极性缺失和/或粘附性下降及其它临床病理学指标(性别、年龄、癌的大小、甲状腺外浸润、淋巴结转移等)的关系。3.设计能够扩增periostin cDNA全长的几对引物,通过RT-PCR法获得扩增的目的DNA片段,并用琼脂糖凝胶跑电泳。将同一胶上不同长度的DNA目的片段分离,纯化,用DNA测序法检测periostin isoform在甲状腺癌组织、非癌组织及癌细胞株中的表达情况,并检测periostin isoform的表达方式与甲状腺癌的发生或癌的进展有无关联。4.对181例有临床预后资料的甲状腺乳头状癌患者(癌直径＜10 mm或手术时有远处转移的除外)进行回顾性分析,调查手术时患者的基本情况以及癌的临床病理学指标,包括性别、年龄、癌的大小、有无甲状腺外浸润、淋巴结转移、pT(UICC的TNM分类)、stage grouping(UICC的TNM分类)、手术断端有无癌等。阅览手术切除标本的HE切片,观察浸润前沿部癌细胞极性缺失和/或粘附性下降的情况。用Kaplan-Meier法和log-rank检验检测浸润前沿部癌细胞极性缺失和/或粘附性下降的情况对患者无病生存率的影响,以及各个临床病理学指标在单因素生存分析中的意义。对在单因素分析中有显著性统计学意义的因素用Cox比例风险回归模型进行多因素生存分析。结果1.68例甲状腺乳头状癌中,浸润前沿部癌细胞极性缺失和/或粘附性下降阳性的病例为42例(61.8%)。癌细胞极性缺失和/或粘附性下降与癌的甲状腺外浸润(P=0.0085),pT高值(P=0.0437)及淋巴结转移(P=0.0015)呈正相关:与患者的年龄,性别及肿瘤大小之间无统计学意义(P＞0.05)。2.periostin的表达用T/N比表示,以T/N=4为临界值,在68例甲状腺癌患者中有26例(38.2%)患者T/N＞4,有42例(61.8%)患者T/N≤4。Periotin的高表达(T/N＞4)与癌浸润前沿部癌细胞极性缺失和/或粘附性下降有显著统计学意义(P＜0.0001):Periotin的高表达(T/N＞4)与癌的甲状腺外浸润(P=0.0005),pT高值(P=0.0015)及淋巴结转移(P=0.0246)呈正相关。3.在人体甲状腺非癌组织、乳头状癌组织、滤泡癌组织、未分化癌组织、乳头状癌培养细胞、滤泡癌培养细胞及未分化癌培养细胞中,periostin isoform的表达方式相同,都有8个isoform被表达。其中5个isoform已被报道,3个是新发现。这8个isoform与野生型periostin相比,在外显子ⅩⅦ～ⅩⅪ中有选择性序列缺失,但这些序列缺失并未引起读码框的移动。3个新isoform的特点分别为外显子ⅩⅦ+ⅩⅧ,ⅩⅦ+ⅩⅪ,ⅩⅦ+ⅩⅧ+ⅩⅨ全部核苷酸序列的缺失。它们的cDNA序列已被基因库(Genbank)收录,编码为:EU262883,EU262884和EU262886。3个新isoform中有两个之前未在任何种类和任何组织中被报道。4.在181例甲状腺乳头状癌患者中,癌浸润前沿部癌细胞极性缺失和/或粘附性下降的阳性率为71.0%,他们的随访时间为131±47(mean±s.d.)月。Kaplan-Meier生存曲线及log-rank检验显示浸润前沿部癌细胞极性缺失和/或粘附性下降的出现与不良临床预后成正相关(P=0.0229)。同时,结果1中浸润前沿部癌细胞极性缺失和/或粘附性下降与癌的甲状腺外浸润,pT高值及淋巴结转移的关系用这批病例得到了又一次验证。5.在单因素分析中,浸润前沿部癌细胞极性缺失和/或粘附性下降、患者年龄、肿瘤大小、甲状腺外浸润、淋巴结转移、pT、stage grouping及手术断端是否阳性对患者的无病生存有显著性影响:在多因素分析中,只有患者年龄和肉眼淋巴结转移是影响患者无病生存的独立变量。结论1.在人体甲状腺乳头状癌中,作为癌上皮细胞间质转化形态学特点的浸润前沿部癌细胞极性缺失和/或粘附性下降的出现与参与癌上皮细胞间质转化过程的分子periostin的高表达有显著的统计学相关性。浸润前沿部癌细胞极性缺失和/或粘附性下降的出现与癌的甲状腺外浸润,pT高值及淋巴结转移呈正相关。生存分析显示,浸润前沿部癌细胞极性缺失和/或粘附性下降阳性的患者其无病生存率明显低于阴性患者。本研究有效地阐述了甲状腺乳头状癌中periostin的表达,浸润前沿部癌细胞极性缺失和/或粘附性下降这一形态学表现,以及患者的临床预后三者之间的相关性。2.人体甲状腺非癌组织、乳头状癌组织、滤泡癌组织、未分化癌组织中,不管是否有甲状腺外浸润或淋巴结转移,periostin isoform的表达方式相同,均有8个isoform同时被表达。这说明periostin isoform的表达方式与甲状腺癌的发生或癌的进展无关。3.本实验发现了人体组织中3个新的periostin isoform,其中有两个未在其他任何生物中报道。更多还原

【Abstract】 Background and objectivePapillary thyroid carcinoma（PTC） is the most common malignancy of the endocrine organs.It is well known that PTC has a good prognosis with a 10-year disease-free survival rate of more than 90%,but still about 10%of patients with PTC develop cancer recurrence or metastasis to the lymph nodes and/or distant organs after surgery and cancer death may occur.Previous studies have revealed that extrathyroid invasion at surgery is significantly correlated with PTC recurrence. Therefore identification of the molecular changes responsible for PTC development and invasiveness could be of great therapeutic significance for PTC treatment.Recently,a novel gene periostin,also called osteoblast specific factor 2（OSF-2） was shown by many authors to have a role in tumor growth,angiogenesis, invasiveness,and metastasis.The up-regulation of periostin promoted tumor progression and angiogenesis through the up-regulation of vascular endothelial growth factor 2 expression in human breast cancers in a cultured cell experiment and tumor xenograft analysis in mice.Non-small cell lung carcinoma patients with high serum periostin levels had significantly poorer survival than the patients with low serum levels.High expression of periostin was reported to be correlated with extrathyroid invasion,lymph node metastasis and higher grade staging in papillary thyroid carcinonomas.Most importantly,transduction of the periostin gene into 293T cells induced cell invasive activity through epithelial-mesenchymal transition （EMT）.The expression of periostin is strikingly up-regulated in the EMT phenotype than in the epithelial phenotype of the squamous carcinoma cells lines.EMT is a common event in PTC invasion,which features by a loss of epithelial properties and the acquisition of mesenchymal properties,including the loss of apical-basal polarity, loss of cell-cell adhesion,loss of E-cadherin expression,and overexpression of vimentin,epidermal growth factor receptor（EGFR）,matrix metalloproteinase-9, TGF-β,NF_kβ,and integrin pathway members in the invasive front.However the survival impact of the loss of cellular polarity/cohesiveness and its relationship with the clinical parameters in PTC such as the status of extrathyroid invasion and lymph node metastasis,and whether periostin expression plays a role in the development of PTC remains unclear.By now,five isoforms of human periostin（h-periostin） with available sequence have been reported from the literature.These isoforms differ at cDNA exonⅩⅤ～ⅩⅩⅢ（corresponding to C-terminus of periostin protein）,due to the alternative splicing in the mRNA level.There is one report describing the difference in the expression pattern of h-periostin between the bladder carcinoma tissue and the non-neoplastic tissue,which indicated that expression pattern of h-periostin was involved in the bladder carcinogenesis.The expression of each h-periostin isoform is totally unknown either in the normal thyroid tissue or in the tumor tissue.This information led us to examine the expression pattern of all h-periostin isoforms in the normal thyroid gland and thyroid carcinoma tissue.Our purpose is to solve the following questions:（1） What is the clinical correlations and survival impact of the loss of cellular polarity/cohesiveness in the invasive front of PTC?（2） What is the clinicopathological significance of differences in periostin expression in tumor and adjacent non-neoplastic tissue?（3） How about the expression pattern of h-periostin isoform in thyroid carcinoma tissue and non-neoplastic tissue? Does the expression pattem of h-periostin in thyroid correlated with carcinogenesis or tumor development?Methods1.68 cases of surgically resected papillary thyroid carcinoma tissues and adjacent non-neoplastic tissues were fixed in 4%formalin and embedded in paraffin for HE（hematoxylin and eosin） section preparation and histological examination. The presence of the loss of cellular polarity/cohesiveness in the invasive front was examined,and was correlated with the other colinicopathological paramenters,such as sex,age,tumor size,extrathyroid invasion,and lymph node metastasis,by using theχ² square test or Fisher’s exact test as appropriate.2.Extract the total RNA from the 68 cases of paired carcinoma tissue and non-neoplastic tissue described above,synthesize the first strand cDNA,and examine the expression of periostin by RT-PCR method.Examine the quantitative expression of periostin by real-time PCR method,using the Taqman probes and primers.β-actin served as an endogenous control.Differences between the matched tumor and non-neoplastic thyroid tissue were calculated using the formula 2exp(ΔCt_tumor -ΔCt_normal) and expressed as a fold change in expression:T/N ratio（T:tumor tissue;N:non-neoplatic tissue）.The correlations between periostin expression and the loss of cellular polarity/cohesiveness and other clinicopathological parameters were calculated by using theχ² square test or Fisher’s exact test as appropriate.3.Design a group of primer sets to amplify the full length of periostin cDNA sequence.RT-PCR was performed to obtain the purpose DNA products.The DNA products were run on agarose gel,isolated from the agarose gel,purified and submitted to direct DNA sequence analysis.The expression pattern of h-periostin isoforms was thus elucidated,and its correlation with thyroid carcinogenesis and tumor development was examined.4.A retrospective study of 181 patients with primary papillary thyroid carcinoma was performed to determine the survival impact of the loss of cellular polarity/cohesiveness in the invasive front.All patients had well-known follow-up data and clinical information at surgery,such as sex,age,tumor size, extrathyoid invasion,lymph node metastasis,pT（TNM classification of UICC）, stage grouping（TNM classification of UICC）,and surgical margins.Their HE sections of surgically resected materials were reviewed to determine the presence of the loss of cellular polarity/cohesiveness in the invasive front.The Kaplan-Meier method and the log-rank test were used to detect the impact of the loss of cellular polarity/cohesiveness in the invasive front on disease-free survival and the significance of every clinicopathological parameters in the univariate analysis.The parameters shown as significant in the univariate analysis were submitted to the multivariate analysis by using the Cox proportional hazards regression model. Results1.In 68 cases of common-type papillary thyroid carcinoma,the loss of cellular polarity/cohesiveness was identified in 42 cases（61.8%）.It was significantly correlated with extrathyoid invasion（P=0.0085）,high pT stage（P=0.0437）, and lymph node metastasis（P=0.0015）,but not statistically correlated with patient age,sex,and tumor size（P > 0.05）.2.Periostin expression was expressed as T/N ratio.If expression of periostin increased more than fourfold,it was considered significant.There were 26 out of 68 patients showing up-regulation of periostin（T/N ratio > 4）.Up-regulation of periostin had a markedly significant correlation with the loss of cellular polarity/cohesiveness in the invasive front（P < 0.0001）.There were also significant positive correlations between up-regulation of periostin and extrathyroid invasion（P = 0.005）,high pT stage（P = 0.0015）,and lymph node metastasis（P=0.0246）.3.The expression pattern of h-periostin isoforms was same in all the samples examined,including papillary thyroid carcinoma tissues and cell lines,follicular thyroid carcinoma tissues and cell lines,and anaplastic thyroid carcinoma tissues and cell lines,that eight h-periostin isoforms were co-existed in every sample.Among the eight isoforms,three were novel findings in the human tissue,and two have not been reported in any species.Compared with the wild type periostin,the three novel isoforms was featured by loss of cDNA sequence in the exonⅩⅦ+ⅩⅧ,ⅩⅦ+ⅩⅪ,ⅩⅦ+ⅩⅧ+ⅩⅨ,respectively. Their cDNA sequences were enrolled by Genbank with accession numbers of EU262883,EU262884,and EU262886,respectively.The deletion of certain cDNA sequence is impossible to cause shift of opening reading frame.4.In 181 patients suffering from common-type papillary thyroid carcinoma,the loss of cellular polarity/cohesiveness in the invasive front was positive in 129 patients（71.0%）.Their follow-up time was 131±47（mean±s.d.） months. There was a significant positive correlation between the presence of the loss of cellular polarity/cohesiveness and poor disease-free survival（P = 0.0229）,by using the Kaplan-Meier method and the log-rank test.Furthermore,the significant correlations between the loss of cellular polarity/cohesiveness and extrathyroid invasion,high pT stage,and lymph node metastasis in Result 1 were confirmed by using this cohort of patients.5.Among all the parameters we examined,the loss of cellular polarity/cohesiveness in the invasive front,age,extrathyroid invasion,pT,gross lymph node metastasis,histological lymph node metastasis,stage grouping,and surgical margin were found to be significant risk factors in univariate analysis. Subsequent multivariate analysis revealed that advanced age（≥60 years） and the presence of gross lymph node metastasis were independent predictors of recurrence;there was no such association for the loss of cellular polarity/cohesiveness in the invasive front.Conclusions1.In human papillary thyroid carcinomas,the loss of cellular polarity/cohesiveness as a histological feature of EMT and the up-regulation of periostin responsible for EMT had a significant correlation.There were also significant correlations between the loss of cellular polarity/cohesiveness and extrathyroid invasion, high pT stage,and lymph node metastasis.Furthermore,survival analysis revealed that the PTC patients with positive loss of cellular polarity/cohesiveness in the invasive front had significant poorer disease-free survival than the patients without.This study was a successful elucidation of a gene expression-histological phenotype-patient outcome correlation.2.The expression pattern of h-periostin isoforms was same in non-neoplastic thyroid tissues,papillary thyroid carcinoma tissues,follicular thyroid carcinoma tissues,and anaplastic thyroid carcinoma tissues,whether there was extrathyroid invasion or lymph node metastasis or not,which indicates that the expression pattern of h-periostin isoforms was correlated with neither thyroid carcinogenesis nor cancer development.3.We identified three novel h-periostin isoforms in the thyroid tissues.Two of them have not previously been reported in any species.更多还原