【作者】 廖佳建；

【导师】 叶长生；

【作者基本信息】 南方医科大学 ， 外科学， 2010， 硕士

【摘要】 乳腺癌是女性最常见的恶性肿瘤之一,严重威胁女性的身心健康。在许多国家,乳腺癌发病率呈升高趋势,是导致女性肿瘤患者死亡的第二大原因。在欧美国家乳腺癌常发生于绝经后妇女,而我国则常见于绝经前妇女,发病年龄较欧美国家年轻。青年乳腺癌与中老年乳腺癌相比,有其不同的临床生理特点,一般认为青年乳腺癌有较强的侵袭性,病情发展快,腋窝淋巴结转移高,预后不良.其可能原因有：①青年乳腺癌患者的卵巢功能旺盛,血液中内源性雌激含量高,可能是肿瘤恶性程度高、转移早、预后差的影响因素；②青年乳腺癌多处于进展期,肿瘤恶性程度高；③青年乳腺癌肿瘤细胞多处于S分裂期,肿瘤细胞侵袭性强、易转移、发展快、预后差；④青年女性正值生育年龄,合并妊娠、哺乳促进乳腺癌恶化,且肿瘤不易被发现,故影响预后；⑤青年女性腺体致密,且是腺纤维瘤、乳腺增生、乳腺炎的好发年龄,故误诊误治率高；⑥青年女性接受临床检查特别是钼靶X线检查的机会少,故易漏诊,就诊晚。VEGF是目前所知道的最强的直接作用于血管内皮细胞的生长因子,在肿瘤血管生长过程中起关键作用；EGFR具有加快肿瘤细胞增殖和血管再生,减少细胞凋亡的作用,常与肿瘤血管形成和肿瘤的转移有关；Ki-67是全面反映细胞群体增殖活性的客观指标,它的高表达是细胞增殖活跃的重要标记,可反映肿瘤细胞的增殖活性。本研究中我们将重点分析乳腺癌组织中VEGF、EGFR和Ki-67的表达情况及其与TNM临床分期和腋淋巴结转移数目的关系,以期进一步探讨青年乳腺癌临床病理学特征。目的探讨青年乳腺癌患者的临床病理特点及血管内皮生长因子(Vascalia endothelial grouth factor, VEGF),表皮生长因子受体(Epidermal growth factor receptor, EGFR)和非组蛋白核蛋白(Ki-67)的表达情况以及与临床特点和病理学特征的关系。达到对青年乳腺癌认识的加深,争取对青年乳腺癌做到早期诊断,规范治疗方案,重视全身治疗,改善其生存率。方法通过电脑病案数据库检索2005年1月至2008年12月间南方医院住院并经手术治疗的符合入选标准的乳腺癌病例,共计477例。从中选择青年乳腺癌患者标准(1)年龄小于35岁或者为35岁,性别均为女性；(2)我院初治的可手术病历；(3)所以病例均病理诊断为癌；(4)术前未进行放疗,化疗,内分泌治疗等；(5)临床病例和病理资料完整；(6)患者民族均为汉族；(7)腋窝淋巴结清扫个数>10个淋巴结的病例。剔除临床资料不完整、少数民族、术前曾接受放化疗等辅助性治疗、乳腺癌复发病例。所选取的乳腺癌病例全部行HE染色、VEGF,EGFR及Ki-67免疫组化染色,从病理科获得所选病例的病理切片以及石蜡块,选择合适的石蜡块制作免疫组化病理白片。采用Envision试剂盒行二步法免疫组化染色。石蜡切片脱蜡至水,柠檬酸缓冲液微波高档抗原修复25分钟,双蒸水冲洗3次,PBS冲洗3次,每次3分钟；过氧化氢酶阻断液微波低档孵育3分钟,清除内源性过氧化物酶活性,双蒸水冲洗3次,PBS冲洗3次,每次3分钟；滴加VEGF、EGFR和Ki-67一抗,电热恒温培养箱37℃孵育1小时,PBS冲洗3次；滴加酶标羊抗鼠IgG聚合物二抗试剂,培养箱中37℃孵育30分钟,PBS漂洗3次；室温下DAB试剂显色约5分钟(3-10分钟),自来水冲洗后苏木素轻度复染约1分钟,盐酸酒精分化约6秒,酒精脱水、二甲苯透明后中性树胶封固切片。染色可疑者即行重新标记染色。以PBS取代-抗作阴性对照,以已知阳性反应片作阳性对照。根据阳性和阴性对照的显色情况,在确定无假阳性和假阴性的前提下VEGF染色乳腺癌细胞胞浆,呈棕褐色；EGFR染色乳腺癌细胞胞膜和(或)胞浆；Ki-67染色乳腺癌细胞胞核,以胞核内有棕黄色颗粒为阳性细胞,凡胞浆有棕黄色着色的不计入阳性细胞内,而作为阴性处理。低倍镜下分别选取阳性细胞最密集区,然后在高倍视野下计数肿瘤细胞。VEGF阴性为<25%细胞浆内着色,弱表达(细小颗粒状)；25%-50%细胞浆内着色,弱表达(细小与较粗大颗粒状并存)为(+)；51%-75%细胞浆内着色,中等表达(较粗大颗粒状)为(++)；>75%细胞浆内着色,强表达(细胞浆内弥漫着色)为(+++)。EGFR阴性为无阳性结果表达或非特异着色；大于10%的区域有阳性表达,胞膜显色不连续为(+)；大于10%的区域有阳性表达,胞膜显色连续,但未形成完整的细胞膜形态,强度中等为(++)；大于10%的区域有阳性表达,胞膜呈连续强阳性表达,细胞膜形态完整为(+++)。Ki-67当阳性癌细胞计数<10%为阴性,兰10%而≤25%为“+”,>25%而≤50%为“++”,>50%为“+++”,≥10%定为阳性表达,其中之25%定为过度表达。采用SPSS13.0统计分析软件包进行数据处理,组间差异性比较采用四格表或行列表卡方检验,两因素相关性分析采用Spearman相关分析,相关系数用r表示,以P<0.05表示差异具有统计学意义。结果1.TNM分期及病理分型按照AJCC第六版方法,青年组中Ⅰ期乳腺癌为11例,Ⅱ期为39例；Ⅲ及Ⅳ期乳腺癌为17例：中老年组分别是21例、252例、137例。青年组Ⅰ期乳腺癌比例显著高于中老年组(x2=11.741,P=0.001),Ⅱ期(x2=0.256,P=0.613)和Ⅲ、Ⅳ期(x2=1.703,P=0.192)乳腺癌相比,差异无统计学意义。青年组浸润性导管癌46例,浸润性小叶癌8例,髓样癌3例,粘液腺癌7例,导管内癌3例；中老年组浸润性导管癌353例,浸润性小叶癌21例,髓样癌例13,粘液腺癌13例,乳头Paget病4例,小管癌4例及大汗腺癌2例。青年组与中老年组在病理学分型上有明显区别,中老年组以浸润性导管癌居多(x2=12.807,P=0.000),而青年组以浸润性小叶癌(x2=4.689,P=0.048)及其它类型癌居多(x2=7.050,P=0.008),差别具有统计学意义。青年组腋淋巴结转移率68.66%(46/67),中老年组为62.20%(255/410)。其中青年组1-3个淋巴结转移占20.90%,转移淋巴结24个占47.76%,中老年组分别为34.88%、27.31%。在转移淋巴结数目1-3、≥4个组分别比较中,青年组与中老年组有显著差异,青年组淋巴结转移≥4的患者明显多于中老年组(χ2=11.421,P=0.001),后者淋巴结转移3个以下者较多(χ2=5.099,P=0.024)。2.免疫组化显示青年乳腺癌患者VEGF.EGFR和Ki-67阳性率分别为61.19%(41/67)、64.18%(43/67)和73.13%(49/67)。中老年组VEGF.EGFR和Ki-67阳性率分别为41.46%(170/410),40.24%(165／410)和58.78%(241／410)。青年组VEGF(x2=9.089,P=0.003).EGFR(x2=13.416,P=0.000)和Ki-67(x2=4.978,P=0.026)的阳性表达率显著高于后者,差异具有统计学意义。3.青年组和中老年组TNM分期与VEGF表达相关度高(r=0.392,P=0.001：r=0.227,P=0.000),与EGFR亦存在相关性(r=0.444,P=0.000；r=0.274,P=0.000),与Ki-67的相关度亦高(r=0.402,P=0.001:r=0.116,P=0.019)；随TNM分期的增加,青年组与中老年组VEGF的阳性表达率逐渐增高,各组间有显著性差异(χ2=11.914,P=0.003；χ2=21.207,P=0.000)；两组EGFR的阳性表达率亦逐渐增高,各组间差异显著(χ2=13.264,P=0.001；χ2=30.871,P=0.000)；青年组与中老年组Ki-67的阳性表达率也逐渐升高,各组间有显著性差异(χ2=10,837,P=0.004；χ2=18.106,P=0.000)。随着TNM分期增加,三者阳性表达率显著升高。4.青年组与中老年组VEGF表达与腋窝淋巴结转移数目呈正相关(r=0.360,P=0.003;r=0.288,P=0.000),两组EGFR表达与腋窝淋巴结转移数目亦存在相关性(r=0.276,P=0.024:r=0.199,P=0.000),青年组与中老年组Ki-67表达与腋窝淋巴结转移数目也呈正相关(r=0.343,P=0.004；r=0.422,P=0.000)；随者淋巴结转移数目的增多,VEGF的阳性表达率逐渐增高,在青年组与中老年组各组间有显著性差异(χ2=8.678,P=0.013；χ2=34.815,P=0.000)；EGFR的阳性表达率亦逐渐增高,在青年组与中老年组各组间有显著性差异(χ2=6.103,P=0.047；χ2=17.052,P=0.000),Ki-67的阳性表达率也逐渐增高,在青年组与中老年组各组间有显著性差异(χ2=7.985,P=0.018；χ2=83.035,P=0.000)。随着腋淋巴结转移数目的增多,三者阳性表达率显著升高。结论1.青年乳腺癌患者的临床病理及生物学特征的提示,青年乳腺癌患者具有侵袭性强,进展快,复发转移早,预后差的特点；2.青年乳腺癌在病理学表现独特,其侵袭性强,预后差可能与癌组织中VEGF、EGFR和Ki-67的高表达有关；3.测量乳腺癌组织中VEGF、EGFR和Ki-67的表达情况对判断乳腺癌预后及指导治疗有一定的参考意义。青年乳腺癌患者的生物学特征的分布有一定规律可循,可作为制定综合治疗方案的依据,对判定预后有一定指导意义。临床工作中,对具有不良的病理学及生物学特征的青年乳腺癌患者,争取早期诊断,精设计治疗方案,重视全身治疗,可望改善其生存率。对青年乳腺癌的研究中,除加深ER、PR、HER-2等研究外,需加强VEGF、EGFR和Ki-67等作用机制的研究,这对评估预后及指导治疗更有意义。更多还原

【Abstract】 Breast cancer is one of the most common malignant tumors of the wowen, threatening the physical and mental health seriously. In many counties,the incidence rate has the tendency to increase, which would be the second principal cause of cancer death in women worldwide.The patients of breast cancer are mosty of wowen in post-menopause in the west,but in our country are mosty in pro-menopause,which is younger than in the west. Young patients with breast cancer have diffent clinic fearures from old patients,which has easy lymph node metastasis, faster progression of disease and poor prognosis.The first probably reason is the young patients with breast cancer have a good ovaries function which making higher endogenous female hormone,it is the probably reason of early metabasis and poor prognosis.The second reason is the young patients with breast cancer are always be in progression, having higher malignancy degree.The third reason is tumor cells are always in s-dividing phase,which have higher invasion, easy metastasis and poor prognosis.The fourth reason is that young women are being in reproductive age, affiliating of pregnancy,suckling make aggravation,and the tumor is not easy to detection.The fifth reason is that young women have pyknoglandular organ,and is the predilection years of adenofibroma, hyperplasia of mammary glands, mammitis,whica make high rates of misdiagnosis and mistreatment.The sixth reason is that young women have few opportunity to receive clinical examination especially molybdenum target,which making easy missed diagnosis and late visit. VEGF is nowadays as far as known the strongest growth factor which making direct effects on endothelial cells in blood vessels, they are-playing an important role in the period of growth of tumor vessels;EGFR can accelerating the reproducing the tumor cells and revascularization, reducing apoptosis.They are relating the formation of tumor vessels und the divert of tumor; Ki-67 is the objective indicator that full disclosure the proliferative activity of the cell colony, its highly express is an important sign of proliferative active of the cells, it can reflect the proliferative activity of the cell colony. Hence the convey of VEGF, EGFR und Ki-67 in cancer tissues, the relationship from clinical stages of TNM Staging and ALNM, and furtherer discuss the characteristic of clinicopathologia of youth breast cancer are our selected analysis.OBJECTIVETo study the expressions of VEGF, EGFR and Ki-67 in young patients with breast cancer, and the correlation between their expressions and clinicopathological characteristics,so as to helping early diagnosis, specifing herapeutic regimen, thinking highly of healing;improving their survival rate.METHEDSWe retrieved 477 cases with breast cancer who were operated in Nangfang Hospital from January,2005 to December,2008 using the medical record database. We study the cases who were 35 years or under 35 years,and the cases can be operated,and all cases are pathological diagnosis as carcinoma;and who were not treated with chemotherapy,radiotherapy and endocrine secretion before operation, and the cases whose clinical data and pathologic diagnosis data was complete;and all cases were Han people;and had numbers of dissected axillary lymph nodes more than 10.Delete the cases who were recurred or had small number of dissected axillary lymph nodes (less than 10) or minority ethnic group or received with chemotherapy,radiotherapy and endocrine secretion before operation,We got the corresponding paraffin blocks from the department of pathology, then made the immunohistochemical sections by the two-step way of shortcut immuno-chemistry. Firstly, deparaffinaged the paraffin section; secondly, repaired the antigen in citrate buffer solution by microwave 25 minutes;thirdly, used the solution of peroxidase to quench the activity of endogenous peroxidase about 3 minutes in microwave, then washed the sections three times separately by Double distilled water and PBS;fourthly, the solution of antibodies to VEGF, EGFR and Ki-67 was applied and the sections were incubated at 37℃in Electric constant temperature incubator about 1 hours, then washed the sections three times by PBS; fifthly, the solution of Goat anti-rabbit HRP polymer LgG was applied and the sections were incubated at 37℃in Electric constant temperature incubator about about 30 minutes, then washed the sections three times by PBS;sixthly, used the solution of DAB to colourate about 5minutes;At last, mild Staininged with Hematoxylin after washed with water, Hydrochloride-Alcohol differentiation about 6 seconds, after dehydration by alcohol and transparent by xylene,mounted by neutral gum.We kept the solution and reaction condition identical,to ensure the unity and comparability. If the color is doubtful, we would carry out the experiment again.We got PBS instead of antibody for negative control and got the known positive reaction sections for positive control.According to the contrast coloring from negative and positive,on the assumption without false positive and false negative the tinctorial VEGF mammary cancer cell endochylemas are sepia; the tinctorial EGFR mammary cancer cell membranes and (or) endochylemas;the tinctorial Ki-67 mammary cancer cell nucleus, they have buffy pellets as positive cells, all buffy colored endochylemas are not account as positive cells, but negative cells.Select the compact district of positive cells under low power lens and count tumor cells under high power lens. The cytolymph of VEGF negative colored<25%,less control (fine particles); 25%-50% cytolymph colored, less control(fine particles with thick particles) marking(+);51%-75% cytolymph colored, middle control (thick particles) marking(++);>75% cytolymph colored, strong control (full)marking(+++).EGFR negative is control without positive or nonspecific colored;cell membrane colored is nonsequence(+) when more than 10% area has positive control;cell membrane colored is sequence without completely cell membrane is middle(++)when more than 10% area has positive control;cell membrane has sequence strong positive control with completely cell membrane is(+++)when more than 10% area has positive control.Ki-67 is negative, when positive cancer cells counts<10%,≥10% and≤25% is,,+",>25% and <50% is,,++",>50% is,,+++",>10%is positive control, und≥25% is over control.Data analyses were carried out with SPSS13.0 statistical software package. Comparison of inter-group differences were analyzed with the fourfold table or list of chi-square test line;Spearman correlation analysis was used to analyzed the relationship between the two factors.P values<0.05 were considered significantRESULTS1.According to the sixth edition of AJCC on TNM Staging and pathology typing, the young patients were 11 cases in stageⅠ,39 cases in stageⅡ,and 17 cases in stageⅢandⅣ;the control group were 21cases,252cases,137cases.The proportion of young patients in stage I were significantly higher than the old-age-group(χ2=11.741,P<0.01),The proportion of young patients in stage II had no difference const to the stageⅢandⅣ(χ2=0.256,P>0.05;χ2=1.703,P> 0.05).In the group of young patients,There were 46 cases of infitrating ductal carcinoma,8 cases of infiltrating lobular carcinoma,3cases of carcinoma medullare,7cases of mucinous adenocarcinoma.3cases of Intraductal carcinoma; In the contrast,there were 353 cases of infitrating ductal carcinoma,21 cases of infiltrating lobular carcinoma,13cases of carcinoma medullare,13cases of mucinous adenocarcinoma,4cases of Pagets carcinoma of the breast,4cases of duct carcinoma,2cases of apocrine carcinoma.There had obviously difference in pathology typing of the youth and the older,there were more cases of infitrating ductal carcinoma than the youth(P=0.000),but there were more cases of infiltrating lobular carcinoma and odther types in the youth(P=0.030,P=0.008),which had statistics significance。The ALNM rate of the youth Were 68.66%(46/67),the older were 62.20 %(255/410).The one to three ALNM rate of the youth were 20.90%,more than four were 47.76%,and the older were 34.88%、27.31%.contrast to the ALNM of one to three and more than four groups,there had significantly difference between the youth and the older(χ2=5.099,P=0.024;χ2=11.421,P=0.001),According to the more than four ALNM,there are more cases in the youth than the older,and the latter had more cases in the less than three groups.2.By using immunohistochenical method, it showed that in the young patients VEGF positive rate was 61.19%(41/67),EGFR positive rate was 64.18%(43/67), Ki-67 positive rate was 73.13% (49/67).In the older patients VEGF positive rate was 41.46%(170/410),EGFR positive rate was 40.24%(165/410), Ki-67 positive rate was 58.78%(241/410).The positive rate of VEGF, EGFR, Ki-67 were higher than the older patients significantly(P<0.05)3.There was significantly positive correlation between TNM Staging and the VEGF positive rate in the young patients and older patients (r=0.392,P=0.001;r=0.227,P=0.000),and there was significantly positive correlation between TNM Staging and the EGFR positive rate in the young patients and older patients(r=0.444,P=0.000;r=0.274,P=0.000),there was also significantly positive correlation between TNM Staging and the Ki-67positive rate in the young patients and older patients(r=0.402,P0.001;r=0.116, P=0.019);with the increase of the TNM Staging, VEGF positive rate was significantly increased in the young group and older group, and there were significant differences among the three groups (χ2=11.914,P=0.003;χ2=21.207,P=0.000);EGFR positive rate was significantly increased in the young group and older group, and there were significant differences among the three groups(χ2=13.264,P=0.001;χ2=30.871,P=0.000); Ki-67 positive rate was also significantly increased in the young group and older group, and there were significant differences among the three groups(χ2=10.837,P=0.004;χ2=18.106,P=0.000).With the increase of TNM Staging, positive rate in three groups were also rising.4:There was significantly positive correlation between ALNM and the VEGF positive rate in the young patients and older patients(r=0.360,P=0.003;r=0.288, P=0.000),and there was significantly positive correlation between ALNM and the EGFR positive rate in the youth patients and older patients(r=0.276,P=0.024; r=0.199,P=0.000),there was also significantly positive correlation between ALNM and the Ki-67 positive rate in the youth patients and older patients (r=0.343, P=0.004;r=0.422,P=0.000);With the increase numbers of the ALNM,VEGF positive rate was significantly increased in the young group and older group, and there were significant differences among the three groups(χ2=8.678,P=0.013;χ2=34.815,P=0.000);EGFR positive rate was significantly increased in the young group and older group, and there were significant differences among the three groups (χ2=6.103,P=0.047;χ2=17.052,P=0.000);Ki-67 positive rate was also significantly increased in the young group and older group, and there were significant differences among the three groups(χ2=7.985,P=0.018;χ2=83.035, P=0.000).With the numbers of the ALNM increase, positive rate in three groups were also rising.CONCLUSION1.According to distribution of the biologic characteristic young cancer patients, they have the specialties of stronger invasiveness, faster progression, earlier recrudescence diverting, and poorer prognosis.2.For its stronger invasiveness and weaker prognosis, the distinctive manifesting of youth breast cancer, which are probably concerned of the highly express of VEGF, EGFR and Ki-67 in cancer tissues.3.The measurements, which made by the convey of VEGF,EGFR und Ki-67 in cancer tissues, have certain references for the prognosis and directed therapy of breast cancer.The distribution of the biologic characteristic young cancer patients has some regular signs, which can be used as basis as multidisciplinary treatment protocols that has certain significance of predicating prognosis.The young patients, who have bad characteristic of pathobiology and biology, should have earlier diagnosis, reasonable treatment prescriptions, and systemic therapies by the clinical work, which can make better survival rate.In the researching of case of youth breast cancer, it will be more meaningful for the appraising prognosis and directing treatment, when it is not only focus on researching for ER, PR, HER-2 etc, but also the further researching of VEGF, EGFR and Ki-67.更多还原