【作者】 喻林；

【导师】 施达仁； 杨文涛； 陆洪芬； 周晓燕；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 博士

【摘要】 目的1.总结基底样型乳腺癌(basal-like breast carcinoma, BLBC)的临床病理和形态学特征。2.完善乳腺癌免疫组化(immunohistochemistry, IHC)分型标准,评价CK/TN、TN、CK、ER/HER2四种分类方法与乳腺癌预后的关系；探讨各亚型预后上的差异,提供病理诊断、临床治疗和预后判断的依据。3.初步研究中国人群乳腺癌的基因表达谱(gene expression profiling, GEP)特征,筛选部分亚型之间的差异表达基因。4.验证三阴性乳腺癌(triple negative breast cancer, TNBC)的部分差异表达基因,分析其与临床病理参数及预后的相关性,为寻找TNBC特异性的分子标记和探索靶向治疗提供新的线索。材料与方法第一部分1.收集复旦大学附属肿瘤医院病理科1997-2002年具有完整临床病理及随访资料的乳腺癌石蜡标本543例,IHC检测ER、PR、HER2、CK5/6、CK14、CK17、EGFR、SMA、P63、vimentin的表达,运用CK/TN、TN、CK、ER/HER2四种分类方法进行IHC分型。2.基底样型乳腺癌的临床病理分析：总结基底样型乳腺癌的临床病理、组织形态学特点；评价基底样和肌上皮标记在基底样型乳腺癌中的表达情况；生存分析临床病理参数、形态学和IHC指标在评估基底样型乳腺癌预后中的意义。3.生存分析比较各亚型乳腺癌预后上的差异及CK/TN、TN、CK、ER/HER2四种分类方法在IHC分型中的差异及其与预后的关系。第二部分1.收集复旦大学附属肿瘤医院2002-2009年乳腺癌新鲜标本及相应石蜡组织50例,IHC检测石蜡组织中ER、PR、HER2、CK5/6、CK14、CK17、EGFR的表达,依据CK/TN分类将50例标本分为5个亚型：腔面A型、腔面B型、HER2过表达型、基底样型和裸表达型。2.50例乳腺癌新鲜标本抽提总RNA,运用琼脂糖凝胶电泳和Alignent2100生物分析仪进行RNA的质量检测。3.运用人类全基因组基因芯片技术对50例乳腺癌进行基因表达谱研究,层次聚类分析完成GEP分型,初步探讨中国人群的基因表达谱特征；筛选部分亚型之间的差异表达基因,并进行差异基因的Pathway和Gene Ontology(GO)分析,初步探讨差异基因的功能。第三部分1.收集复旦大学附属肿瘤医院2002-2009年乳腺癌新鲜标本92例(三阴性42例,腔面型50例),运用实时荧光定量逆转录聚合酶链反应(Real-time quantitative reverse transscripton polymerase chain reaction, Real-time RT-PCR)技术检测92例乳腺癌中SLC6A14、PTX3、FOXC1mRNA的表达,分析其在三阴性和腔面型乳腺癌之间的表达差异,以验证第二部分基因芯片的检测结果,并进一步分析SLC6A14、PTX3、FOXC1mRNA表达与临床病理参数的相关性。2.另收集1997-2003年具有完整临床病理资料及随访资料的乳腺癌石蜡包埋组织94例(三阴性57例,腔面型37例),IHC检测FOXC1蛋白的表达,比较FOXC1蛋白在三阴性和腔面型乳腺癌之间的表达差异,并分析其与临床病理参数及预后的相关性。结果第一部分1.依据CK/TN分类的IHC分型结果：基底样型乳腺癌占总例数的19.9%,腔面A型、腔面B型、HER2过表达型和裸表达型分别占42.2%、10.5%、16.6%、10.9%。2.基底样型乳腺癌的临床病理特征：发病年龄31-91岁,平均52.4岁,与其它各亚型无明显差异(P值>0.05)。肿瘤平均最大径2.9cm,显著大于腔面A型(P<0.05),与其它亚型无明显差异。淋巴结转移率为47.6%,低于腔面A型、HER2过表达型和裸表达型。75.1%的BLBC组织学分级为3级,显著高于非基底样亚型(P值均<0.001)。3.基底样型乳腺癌的组织病理学特征：70.4%的BLBC呈推挤性生长,72.2%以弥漫实性片状结构为主；67.3%可见合体细胞；61.1%的BLBC见泡状核,58.3%具有明显的核仁；BLBC核分裂计数3级占74.1%；化生的发生率为21.3%；73.1%伴间质淋巴细胞浸润；以上各特征在BLBC中的发生率显著高于其它各亚型(P<0.01)。BLBC坏死率高,以大片地图样坏死(38.0%)和中央性坏死(24.1%)为主。大片地图样坏死的发生率显著高于腔面型及HER2过表达型(P<0.01)。4.基底样型乳腺癌中基底及肌上皮标记的表达：基底样标记CK5/6阳性率最高,达63.0%,其余依次为CK17 (49.1%)、CK14 (47.2%)、EGFR (37.0%)。肌上皮标记的阳性率为23.1%。5.生存分析(1)543例乳腺癌的无病生存率和总生存率分别为61.7%、80.8%。局部复发率、远处转移率、病死率分别为9.8%、34.8%、19.2%。(2)裸表达型复发率最高(18.6%),其次为基底样型(12.0%),裸表达型的复发率显著高于腔面A型(P<0.05)。HER2过表达型转移率最高(46.7%),其次为基底样型(45.4%),基底样型、HER2过表达型和裸表达型的转移率均显著高于腔面型(P<0.05)。基底样型肺、脑转移率分别为22.2%、10.2%,高于其它各亚型,与腔面A型差异有显著性(P<0.05)。腔面A型骨转移率最高(39.3%),高于其它各亚型。(3) BLBC中,高临床分期组、淋巴结阳性组、出现脉管侵犯组、CK5/6阳性组的总生存率及无病生存率分别显著低于低临床分期组、淋巴结阴性组、无脉管侵犯组、CK5/6阴性组(P<0.05)；核分裂计数3级组的总生存率显著低于核分裂计数2级组(P<0.05)。呈膨胀性生长、伴淋巴细胞浸润的BLBC的总生存率及无病生存率分别高于呈浸润性生长、不伴淋巴细胞浸润的BLBC,差异具有显著性(P<0.05)。淋巴细胞浸润,CK5/6表达是BLBC独立的预后因素。6.四种分类方法对各亚型预后判断结果的比较(1)对无病生存率的判断结果：CK/TN、TN和ER/HER2分类对各亚型无病生存率的评价结果基本相同。而采用CK分类,腔面B型无病生存率显著高于基底样型,基底样型显著低于裸表达型。(2)对总生存率的判断结果：四种分类方法相同的结果是,腔面A型总生存率显著高于非腔面型。而对其它各亚型的判断存在一些差异。1.RNA质检：所有样本RNA的纯度和完整性良好,符合表达谱芯片的要求。2.聚类分析：(1)50例样本大致可分为以腔面A型、HER2过表达乳腺癌、三阴性乳腺癌为主的三个亚型；HER2过表达型与腔面B型、基底样型与裸表达型显示相似的基因表达谱特征。(2)IHC分型界定的三阴性、非三阴性、腔面A型乳腺癌与GEP分型能较好的对应吻合,但在其它亚型的界定上存在重叠交叉。3.差异表达基因的筛选：腔面A与B型的差异基因共190个,均表达上调；腔面A与HER2过表达型的差异基因共137个,73个基因表达上调,64个表达下调。腔面B与HER2过表达型的差异基因仅8个,均表达上调；三阴性与腔面型的差异基因共611个,217个基因表达上调,394个表达下调；三阴性与非三阴性乳腺癌的差异基因共411个,167个基因表达上调,244个基因表达下调。4. TNBC与腔面型乳腺癌差异基因的Pathway和GO分析,差异基因参与了多条通路和多种生物学过程。第三部分1. Real-time RT-PCR检测结果与基因芯片检测结果对比：SLC6A14、PTX3、FOXC1在TNBC和腔面型乳腺癌中差异表达趋势一致。2. SLC6A14、FOXC1mRNA在TNBC中表达显著高于腔面型(P<0.05)。PTX3两者之间的表达无显著差异(P>0.05)。3. SLC6A14、PTX3、FOXC1mRNA表达与三阴性乳腺癌肿瘤大小、组织学分级、临床分期、淋巴结转移以及患者年龄均未见相关性(P>0.05)。4. FOXC1蛋白主要定位于细胞浆、可见于细胞膜,正常乳腺上皮和肌上皮FOXC1蛋白均为强阳性染色。FOXC1蛋白在TNBC中的阳性率为66.7%,基底样型乳腺癌中的阳性率为76.6%,腔面型中的阳性率为32.4%,三阴性及基底样型乳腺癌中FOXC1蛋白的表达水平显著高于腔面型(P=0.002)。5.三阴性乳腺癌中高年龄组(>50岁)与低年龄组(≤50岁),FOXC1蛋白表达有显著差异(P<0.05)。FOXC1蛋白表达与肿瘤大小、组织学分级、临床分期、淋巴结转移均未见相关性(P>0.05)。6.三阴性乳腺癌中FOXC1阳性组的无病生存率和总生存率均低于FOXC1阴性组,但两者差异无显著性(P>0.05)。结论1.基底样型乳腺癌具有独特的临床病理和形态学特征；CK5/6是其最敏感的标记,CK14特异性最强。2.临床分期、淋巴结状态、膨胀性生长、淋巴细胞浸润、脉管侵犯、核分裂计数、CK5/6表达与基底样型乳腺癌临床预后相关。高临床分期、淋巴结阳性、脉管侵犯、高核分裂计数、CK5/6阳性提示预后差；膨胀性生长、淋巴细胞浸润提示预后好；淋巴细胞浸润、CK5/6是基底样型乳腺癌的独立预后因素。3.各亚型乳腺癌的无病生存率、总生存率、复发率、转移率及转移部位分布存在一定差异。4. CK/TN、TN、CK和ER/HER2分类在评价不同亚型乳腺癌的预后方面存在差异。5.中国人群乳腺癌GEP分型可能不同于西方人群,其具有自身的特点：分型相对简单,可分为三个亚型,腔面A型、HER2过表达乳腺癌、三阴性乳腺癌；HER2过表达型与腔面B型、基底样型与裸表达型显示相似的基因表达谱特征,可能属于同一个分子亚型。6.当前使用的乳腺癌免疫组化分型方法仍存在一定的局限性,在部分亚型的界定上与GEP分型不能很好的对应和吻合。7.经基因芯片检测分析,部分亚型乳腺癌之间基因表达存在明显差异,差异基因参与了不同的Pathway和GO分类,提示各亚型乳腺癌可能存在不同的发生发展机制。8. TNBC中SLC6A14、PTX3、FOXC1mRNA的表达水平高于腔面型,它们可能与TNBC的发生发展有一定的相关性。9. TNBC及BLBC中FOXC1蛋白的表达显著高于腔面型,其可望成为TNBC、BLBC新的特异性分子标记。更多还原

【Abstract】 Objective:To summarize clinicopathological and morphological features of basal-like breast carcinoma (BLBC); To perfect the standards of immunohistochemistry (IHC) classification in breast cancer. To evaluate the prognostic differences among subtypes of breast cancer using four IHC classifications (CK/TN, TN, CK, ER/HER2), and guide clinical treatment and prognosis evaluaton; To study GEP features of breast cancer in Chinese people and screen differentially expressed genes among subtypes of breast cancer; To validate several differentially expressed genes of triple negative breast cancer (TNBC) and analyze their correlation with clinicopathological parameters and prognosis, and provide new clue of looking for specific molecular markers and targeted therapy of TNBC.Materials and methods:PartⅠ:(1) The formalin-fixed paraffin-embedded samples of 543 cases of breast cancer with complete clinicopathologic and follow-up data were collected in the Department of Pathology, Cancer Hospital, Fudan University. The cases were initially diagnosed between 1997 and 2002. Immunohistochemical staining of ER, PR, HER2, CK5/6, CK14, CK17, EGFR, SMA, P63, vimentin was performed. Four classifications of CK/TN, TN, CK, ER /HER2 were used to IHC phenotyping analysis. (2)The clinicopathological and morphological characteristics of BLBC were summarized; the expression of basal-like and myoepithelial markers were analyzed in BLBC. (3) The relationship between clinicopathological, morphological, immunohistochemical parameters and prognosis in BLBC was investigated by survival analysis. The prognostic differences of breast cancer subtypes were compared using CK/TN, TN, CK, ER/HER2 classifications.PartⅡ:(1) The frozen tissues and corresponding formalin-fixed paraffin-embedded samples of 50 cases of breast cancer from 2002-2009 were obtained from Cancer Hospital, Fudan University. Immunohistochemistry study of ER, PR, HER2, CK5/6, CK14, CK17 and EGFR was performed. According to CK/TN classification, the cases were divided into five subtypes:luminal-A, luminal-B, HER2-overexpression, basal-like and null subtype. (2)The total RNA of 50 frozen samples was extracted and purified, and RNA quality was controlled by agarose gel electrophoresis and Alignent 2100 bioanalyzer. (3)Gene expression profiling of 50 cases of breast cancer was studied using genomewide microarray technology. The GEP classification was completed by hierarchical clustering analysis, to study GEP characteristics of Chinese breast cancer. Differentially expressed genes between different subtypes were screened and their biological functions were analyzed by Pathway and Gene Ontology (GO) analysis.PartⅢ:(1) The frozen tissue samples of 92 cases of breast cancer acquired from 2002-2009 were collected in Cancer Hospital, Fudan University. The expression of SLC6A14, PTX3, FOXC1 mRNA in 92 cases were detected by Real-time RT-PCR, and the expression of these genes was compared between TNBC and luminal subtype to validate the results of microarray. (2) The formalin-fixed paraffin-embedded samples of 94 cases of breast cancer with complete clinicopathologic and follow-up data acquired from 1997 to 2003 were collected, including BLBC (57 cases) and luminal subtype (37 cases). The expressin of FOXC1 protein was detected by IHC staining, to compare its expression difference between TNBC and luminal subtype and study its association with clinicopathologic parameters and prognosis in breast cancer.Results:PartⅠ:(1) According to CK/TN classification, BLBC, luminal-A, luminal-B HER2-overexpression and null subtype constituted 19.9%,42.2%,10.5%,16.6%,10.9% of breast cancer, respectively. (2) The average tumor size was 2.9cm, significantly bigger than luminal-A (P>0.05). The metastasis rate of lymph node was 47.6%, lower than luminal-A, HER2-over expression and null type.75.1% of BLBCs were histologically grade 3, higher than other non-basal-like subtypes. (3) The histopathologic features of BLBC: 70.4% of BLBCs showed expansive growth,72.2% showed diffusely solid pattern; Syncytial cells, vesicular nuclei and prominent nucleoli, nuclei of grade 3 and metaplasia was seen in 67.3%,61.1%,58.3%,74.1%,21.3% of BLBC, respectively. The necrosis rate of BLBC was high and predominant of geographic and central necrosis. The incidence of geographic necrosis was significantly higher than luminal and HER2-overexpression type. (4)The expression rate of CK5/6 was highest among basal-like markers, up to 63.0%. The expression rate of CK17, CK14, EGFR was 49.1%,47.2%,37.0%, respectively. The expression rate of myoepithelial markers was 23.1%. (5) Survival analysis:(a) The disease-free survival and overall survival of 543 cases were 61.7% and 80.8% respectively. Local recurrence, distant metastasis and fatality rate were 9.8%,34.8%,19.2%, respectively. (b)The recurrence rate was highest in null type (18.6%), the second was BLBC. Pulmonary and brain metastasis rate of BLBC were 22.2% and 10.2% respectively, higher than other subtypes. (c) Univariate analysis demonstrated the following as poor prognostic factors for overall and disease-free survival:high clinical staging, positive lymph node, vascular invasion, positivity with CK5/6; the following as good prognostic factors for overall and disease-free survival:expansive growth and lymphocytic infiltrates. Multivariate analysis indicated that lymphocytic infiltrates and expression of CK5/6 were independent prognostic factors of BLBC. (3) The evaluation results of prognosis were essentially consistent using CK/TN, TN, ER/HER2 classifications, but CK classification was different from other three classifications.PartⅡ:(1) The purity and integrity of RNA in all samples was good. (2) 50 cases of breast cancer can be divided into three groups predominant of luminal-A, HER2-overexpression breast cancer (including HER2-overexpression subtype and luminal-B) and TNBC. There was similar expression pattern between HER2-overexpression and luminal-B, basal-like and null subtype. (3) TNBC, non-TNBC, luminal-A subtype defined by IHC was consistent with GEP classification, there was certain overlapping in distinguishing luminal-A and luminal-B, luminal-B and HER2-overexpression type, HER2-overexpression and TNBC, basal-like and null subtype.(4)Screening of differentially expressed genes showed that the number of differentially expressed genes of luminal-A vs. luminal-B subtype, luminal-A vs. HER2-overexpression subtype, luminal-B vs. HER2-overexpression subtype, TNBC vs. non-TNBC, TNBC and luminal subtype was 190,137,8,611,411, respectively.PartⅢ:(1) The expression of SLC6A14, PTX3 and FOXC1 mRNA in TNBC and luminal subtypes of breast cancer is consistent in Real-time RT-PCR and microarray detection. (2) The expression of SLC6A14 and FOXC1 mRNA in TNBC was significantly higher than luminal subtype of breast cancer. There was no significan difference of PTX3 mRNA expression between TNBC and luminal subtype of breast cancer. (3) No relationship was found between SLC6A14, PTX3 and FOXC1 mRNA expression in TNBC and other clinicopathologic factors of such as tumor size, histological grade, clinical stage, lymph node metastasis and age of patients. (4) FOXC1 protein was located in the cytoplasm and was occasionally seen in the cell membrane. The normal mammary epithelium and myoepithelium were strongly positive for FOXC1 protein. The epresssion rate of FOXC1 protein in TNBC and BLBC was significantly higher than luminal subtype of breast cancer (66.7% vs.32.4%; 76.6% vs.32.4%). (5) There was significant difference of FOXC1 protein expression between the high age group and low age group. No relationship was found FOXC1 protein expression and other clinicopathologic parameters such as tumor size, histological grade, clinical stage, lymph node metastasis in TNBC. (6) The disease-free and overall survival of FOXC1-positive group was lower than FOXC1-negative group, but there was no statistical significance.Conclusions:(1) BLBC shows distinctive clinicopathological and morphological features; CK5/6 is most sensitive of all basal-like markers and the specificity of CK14 is highest. (2)Clinical stage, metastasis of lymph node, expansive growth, lymphocytotic infiltrates, high mitotic index and expression of CK5/6 are associated with prognosis of BLBC. (3)There are some disparities in recurrence and metastasis rate, and distribution of metastasis sites between different subtypes of breast cancer. (4)CK/TN, TN, CK and ER/HER2 classifications have certain differences on evaluation of the prognosis of each subtype of breast carcinoma. (5) SLC6A14 and FOXC1 mRNA expression in TNBC is significantly higher than luminal subtype of breast cancer. There is possibly an association between these genes and developing of TNBC. (6)FOXC1 protein expression in TNBC and BLBC is significantly higher than luminal subtype of breast cancer. It is promising to become a specific molecular marker of TNBC and BLBC.更多还原