【作者】 刘万军；

【导师】 范志民；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 三阴乳腺癌(Triple negative breast cancer, TNBC)是最近几年被提出来的一种乳腺癌亚型,专指雌激素受体(estrogen receptor, ER)、孕激素受体(progesterone receptor,PR)和人类表皮生长因子受体2(human epidermal growth factor receptor 2, HER2)都是阴性的乳腺癌。此类乳腺癌具有特殊的生物学行为和临床病理特征,表现为侵袭力强、远处转移风险高和预后差等特点。由于三阴乳腺癌的治疗目前属于摸索阶段,尚没有大规模的多中心的临床研究,还没有特有的针对三阴乳腺癌的有效治疗,因而引起医学界的广泛关注。表皮生长因子受体(Epidermal growth factor receptor, EGFR)是一种酪氨酸激酶受体,具有酪氨酸激酶(Tyrosine kinase, TK)活性,存在于细胞膜表面,属于糖蛋白受体,它在正常细胞和恶性肿瘤细胞的生长调控方面起着重要的作用。EGFR可以通过多种机制促进细胞恶性转化,包括受体的过度表达、突变、生长因子受体自分泌环的活化以及特定的磷酸酶失活等。其中涉及肿瘤发生和进展的机制中,最常见的是EGFR的过度表达。研究发现,EGFR在多种肿瘤细胞中呈高表达,特别是在非小细胞肺癌(NSCLC)组织中,EGFR的阳性率可高达80%以上。根据EGFR对细胞生长的调控机制,一些合成药物被研制出来,如吉非替尼、西妥昔单抗、赫赛汀、范得它尼等。这些药物可以阻断EGFR介导的信号传导,从而抑制肿瘤细胞的生长、周期的延长,并抑制肿瘤对周围组织的侵袭及远处转移,促进肿瘤细胞的凋亡。经过近几年的临床实践和疗效观察,这种针对EGFR的靶向治疗在肿瘤治疗中的作用已得到了医学界的广泛肯定。基于TNBC在治疗上的难题,以及EGFR在肿瘤治疗中的突出作用,我们把EGFR和TNBC联系起来,把EGFR在TNBC中的表达以及对TNBC的预后影响作为本论文课题研究的出发点,通过免疫组化法(IHC)、荧光原位杂交(FISH)、聚合酶链反应(PCR)等免疫学及分子生物学等实验方法,探究EGFR在TNBC的表达情况；EGFR表达与TNBC的各临床病理特征之间的联系；EGFR与TNBC预后的相关性。本实验结果显示,EGFR在TNBC中呈高表达,但扩增不明显,且未发现有突变情况；EGFR与TNBC的各病理特征之间未发现有统计学意义的分析结果；EGFR的过表达对TNBC的预后有明显的影响。因此,我们不难得出这样的结论：EGFR可做为评价TNBC预后的一个重要的指标,并可为TNBC提供一个靶向治疗的突破口。更多还原

【Abstract】 The aim of this study was to investigate EGFR expression in TNBC, fund the relationship between the EGFR and the prognosis of TNBC patient, clarify the significance of EGFR in TNBC, give valuable information to guide further studies for the treatment of TNBC.INTRODUCTIONEGFREGFR (Epidermal growth factor receptor) belongs to the HER family, and also known as HER1,is located on 7q12. EGFR is a 170-kDa transmembrane protein with tyrosine kinase activity that upon binding to its ligand. In cellular regulation mechanisms involving proliferation, differentiation and angiogenesis of cells, EGFR has a central position. Although EGFR is important in the maintenance of normal cellular function and survival, EGFR expression clearly appears to contribute to the growth and survival of tumor cells. EGFR expression has been reported to be an indicator of potential tumor progression and/or patient prognosis for such malignant tumors as esophageal cancer, gastric cancer, head and neck cancer, and ovarian cancer. A lot of studies confirm that the EGFR signal transduction pathways have been correlated with various processes that contribute to the development of malignancie. Because of the importance and the role of the EGFR pathways in cell cycle progression and tumor cell proliferation, different strategies were developed to block or downregulate EGFR. These include monoclonal antibodies to the EGFR, tyrosine kinase inhibitors, ligand-linked toxins, and antisense approaches.TNBCTriple-negative breast cancers (TNBC) are defined by a lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression. In 2001, Sorlie and Perou, et al. classified breast tumors into five types, i.e., the Duct A and B, Her-2 overexpression, basal-cell, and normal breast types, after an analysis of the gene expression spectrum. It was shown in the study by Carey, et al. that 80% to 90% of the TNBC cases belonged to the basal-cell type. TNBC generally occurs in younger women, less than 50 years, and is associated with a high risk of death compared with non-triple-negative tumours, regardless of disease stage at diagnosis, particularly in the first 3 years after diagnosis. On average, TNBC show a higher rate of node positivity at the time of diagnosis than other breast cancers. TNBC patients have increased frequency of distant metastasis formation, but not of local relapse. This metastatic progression is marked by early relapse with predominance of visceral and CNS metastases, and lower rate of bone metastases. In the treatment, although many trials had been done, but there is no definitive effect for TNBC all the time.MATERIALS AND METHODSPatientsIn total,572 cases of breast cancer undergoing surgery at First Hospital of Jilin University in China from January 1,2000 to December 31,2005 were informative for ER, PR, and HER2. Of these informative cases,68(11.9%) female patients were found to be TNBCs. For 14 of 68 patients, their chemotherapy after surgery was not determined, a further 6 patients absenced integrated record about their survival data,5 of the remaining patients paraffin-embedded tissues was. insufficient to perform a series of analysis,1 patient absenced integrated record about her clinicopathological. Therefore,42 patients with TNBC were selected as research objects (TNBC group). As reference group, we select randomly 40 female patients which Immunohistochemical marked HER2 grade 3+from 572 cases (HER2 group). Because HER2 protein expression as a classic clinical feature has a important pole in the evaluation of breast cancer, we consider that it is significant to selecting HER2(3+) patients as reference group in this study.MethodsSpecimens were obtained from patients who underwent surgery. All 82 cases were subjected to Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH)and reverse transcription-polymerase chain reaction (RT-PCR) to investigate EGFR gene expression, amplification and mutation. Relationship of EGFR expression and the prognosis of TNBC patient was investigated. Statistical analysis was done using the Statistical Package for Social Sciences, Version 18.0 (SPSS).ResultsTNBC was significantly associated with age (P=0.033). No significant difference was observed regarding such tumor-related factors as tumor size, lymph node metastasis, and tumor stage, menstruation status between two groups.Immunohistochemical study for EGFR expressionAn immunohistochemical overexpression of EGFR was 34 of 82 (43.9%) breast cancer cases. Proportion of EGFR overexpression among triple negative tumors was comparatively higher than that of HER2(3+) (P=0.067),overexpression of EGFR occurring in 24 of 42 (57.1%) patients in TNBC group and 10 of 40 (25.0%) patients in HER2(3+) group. No association between EGFR overexpression and clinicopathological features was observed in two groups.FISH analysis for EGFR gene amplificationWith FISH,5 of 82 specimens could not be evaluated because of uninterpretable results of the hybridization itself. The other 77 cases could be analyzed (TNBC39, HER2(3+)38), of which 7 (9.1%) showed amplification:five triple negative tumours and two HER2 case (ER-/PR-, ER-/PR-). All cases with EGFR gene amplification by FISH showed EGFR overexpression on IHC.PCR and Direct sequencing for EGFR MutationDNA extraction and polymerase chain reaction (PCR) of EGFR (exons 19 and 21) were done among the 82 patients,56 (TNBC 27, HER229) was successful. Direct sequencing analysis revealed no EGFR gene mutation in any of the successfully tested cases (n= 56).Patients survival according to EGFR expressionThe follow-up was discontinued in December 2010. After Kaplan-Meier analysis, the 5-year OS rates were 71.4%and 87.5%, the 5-year DFS rates were 57.1%and 77.5%, respectively in the TNBC and HER2 groups.During the median follow-up period of 82 months range:(12-124 months),50 patients (61.0%) had recurrent disease (TNBC 28, HER222) and 27 patients (32.9%) died of recurrent breast cancer (TNBC 18, HER29). Of 34 patients with EGFR-overexpressing tumors,25 patients (73.5%) had recurrent disease (TNBC 19, HER2 6) and 19 patients (55.9%) died of recurrent disease during the follow-up period (TNBC 15, HER2 4). Of the remaining 48 patients with EGFR-negative tumors,25 patients (52.1%) had recurrent disease (TNBC 9, HER2 16) and 8 patients (16.7%) died of disease during the follow-up period (TNBC 3, HER2 5). Survival analysis was performed on the 82 patients with disease-free survival (DFS)and overall survival (OS), the TNBC patients were decreased when compared to those of the HER2 (3+) patients CP=0.017 for DFS, P=0.019 for OS). Both disease-free survival and overall survival of the patients having EGFR-overexpressiing tumor were decreased when compared to those of the patients with EGFR-negative tumor (P=0.027 for DFS, P=0.038 for OS). In the triple-negative subgroup, the overall survival rate of EGFR-overexpressing patients was statistically significantly lower than that of EGFR-no-overexpressing patients (P=0.018 for DFS, P=0.026 for OS). In the HER-2 subgroup, there was no statistical difference (P=0.079 for DFS, P=0.055 for OS).DiscussionTriple negative breast carcinoma (TNBC) have a higher malignancy, more aggressive invasion, a higher proliferation rate and poorer clinical prognosis compared to other types of breast cancer. The EGFR-mediated signal transduction accounts for one of the most important pathways in carcinogenesis, and the activation of the pathway could promote such biological processes as apoptosis, cellular differentiation, tumor proliferation, invasion, adhesion, and DNA repair. In the previous literature, HER2 gene has been extensively studied in breast cancer, but the data on EGFR gene in breast cancer are limited. In recent years, more and more research about EGFR expression in breast cancer has been investigated in a variety of studies, but the research of EGFR expression in TNBC is rare. In particular, the literature concerning EGFR expression and molecular changes within TNBC tumors is sparse. The aim of this study is investigating the relationship of EGFR expression and the TNBC tumors through analysising data which obtain from immunohistochemistry and FISH and PCR.Compared with non-TN breast cancers, TNBC have an onset at a younger age, a larger mean tumor size, higher grade, higher rate of node positivity, In our study, we only notice the age and nuclear grade have significant difference and the other tumor-related factors have no significant difference between the two groups. A principle explanation for the discrepancies reported to date concerns the various methods used to assess these statuses. Other explanations (and limitations) for our findings are that our sample size was small. Over past few years, it has been shown in studies that there was an up-regulation of the EGFR expression in the TNBC patients, and EGFR overexpression is seen in approximately 45-70%. Our research have a similar finding that the overexpression rate of EGFR was 57.14% in the TNBC group and significantly higher than in the HER2(3+) group(25.0%).In the further study, we found no correlation between EGFR expression levels and clinico-pathological data in two groups.We further investigated the relation between EGFR overexpression by IHC and EGFR gene copy number by FISH in TNBC. In all case, only 7(9.1%) showed gene amplification, much less frequent than HER-1 overexpression 34(41.5%). In another similar study, observations were made by S Pintens et al, reporting 8.8% EGFR gene amplification and 35.1% EGFR overexpression. Comparing our FISH results with that of the former study, both showed a similar percentage of EGFR gene amplification and overexpression. These results suggest that EGFR gene amplification may not be the only mechanism of EGFR protein overexpression in TNBC; there may be other possible mechanisms and pathways that can cause EGFR overexpression. Besides gene amplification, another mechanisms can lead to aberrant EGFR expression in cancer, including receptor overexpression, abnormal mRNA slicing, mutations at gene promoter region and ligand-independent activation. Understanding this mechanism may help to locate novel molecular targets in drug discovery in treatment. Unfortunately, the mechanism of EGFR protein overexpression is poorly understood in TNBC. Notably, in our results,7 positive cases (5TNBC,2HER-2) with by FISH all showed EGFR overexpression on IHC. Because patient numbers (7cases) are too small for statistical analysis, we can’t confirm that EGFR FISH-positive status correlate with EGFR overexpression in breast cancer, especially in TNBC. In more detail, we also observed 7 cases of EGFR gene amplification total with a negative ER status. Whether EGFR gene amplification associated with negative ER in breast cancer, it is necessary to further investigate. We sought mutations in the EGFR gene in TNBCs, because these mutations were suggested to predict responses to gefitinib therapy. In our study, we investigate whether EGFR mutations (exons 19and21) could also be a feature of TNBC. But we did not find any mutations in the tested exons of TNBC and HER2 (3+) breast cancer suggesting that EGFR mutations do not contribute to receptor activation in breast cancer. However, the amount of case (56) is limited, our study cannot exclude the possible presence of a EGFR mutation in a small subset of breast cancer patients. Whether EGFR mutation exist in TNBC and could be a marker of sensitivity to antireceptor therapy, we suggest that it remains to be established in specific clinical trials.Our researches show that disease-free survival (DFS) and overall survival (OS) in TNBC are less than that in HER-2. Moreover, our results indicate that OS and DFS rate of EGFR overexpression patients are worse than EGFR no-overexpression patients in triple-negative breast cancer group, but this observation no be fund in HER2 group. In other words, EGFR overexpression in the triple-negative breast cancer was a poor prognostic factor in our study.Because of the development and technical progress of molecular-targeting drugs for cancer therapy, EGFR signalling has been successfully targeted in many cancer types using humanised anti-EGFR monoclonal antibodies (e.g. cetuximab) and EGFR tyrosine kinase inhibitors (e.g. gefinitib). Some literatures focused using cetuximab or gefinitib as a possible candidate for molecular-targeting on patients with TNBC expressing EGFR. Unfortunately, based on several multicenter clinical researches, the commonly-used molecular target drugs are confirmed ineffective in TNBC. For this phenomenon, Normanno N. consider, although tissues from TNBC patients often express EGFR, EGFR tyrosine kinase inhibitors have little efficacy, which partly arises through intrinsic resistance mechanisms within the tumors. B. Corkery consider, despite high levels of EGFR protein in TNBC cells, they may not be dependent on EGFR signalling for growth. Although the TNBC cells are not inherently sensitive to EGFR inhibition, several clinical trials are currently assessing that combined treatment with gefitinib and chemotherapy have a greater effect on TNBC patients. Moreover, TNBC patients have increased frequency of distant metastasis formation, but not of local relapse, indicating that these tumors are generally sensitive to irradiation. CONCLUSIONIn this study, we documented EGFR overexpression, both EGFR gene amplification and mutation, in triple negative and HER2 (3+) breast patients. We noticed a higher incidence of EGFR overexpression compared to EGFR amplification, and no EGFR gene mutation, in TNBC. However, no correlation between EGFR overexpression and clinico-pathological data. We have found that EGFR is a strong prognostic factor for relapse-free survival and overall survival for the TNBC patients. Under these treatment conditions, overexpression of EGFR could provide information concerning a poor outcome in TNBC.更多还原