乳腺癌新辅助化疗疗效判断及基因表达与预后分析研究
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摘要
美国癌症协会预计2008年美国女性乳腺癌有182460新发病例,40480死亡病例,发病率26%居肿瘤的第一位[1]。尽管亚洲是乳腺癌低发区,但在我国经济发达地区,乳腺癌呈明显的上升趋势,每年新增病例达3-4%,成为发病率上升最快的恶性肿瘤之一,而且有年轻化趋势[2]。人们面临如此庞大的乳腺癌患者,寻找肿瘤相关基因、合理治疗以提高生存率显然是目前亟待解决的问题。乳腺癌患者术前新辅助化疗是临床治疗的一大进展,不但使更多患者获得了手术根治的机会,而且还使不少化疗后病理完全缓解的患者延长了生存。另外,由于分子生物学已深入应用到肿瘤领域的各个方面,乳腺癌也从临床试验为依据的循证医学治疗模式逐渐向更完美的个体化治疗转化。但是目前的个体化治疗仍多基于简单的临床病理特征而制定治疗方案,所达到的效果并不理想,这也是肿瘤内科所面临的重要问题。因此只有真正认识每个个体的肿瘤基因表达情况,才有可能达到有的放矢的治疗,为患者选择合理、有效的方案,避免治疗不足或治疗过度带来的毒副反应和经济负担。本研究课题即围绕上述问题进行了初步探讨。
第一部分对乳腺癌的新辅助治疗中临床存在的一些问题进行了初步探讨。乳腺癌新辅助化疗(NAC)目前已成为乳腺癌的标准治疗,治疗后获得病理学完全缓解(pCR)的患者预后最好,因此在我国也普遍开展。但国外关于NAC疗效评价手段的研究不够详细,国内更是鲜有报道,因此我们主要比较了查体、B超和钼靶在乳腺癌NAC疗效评价中的差异,并分析治疗前后肿瘤特征的变化。方法是对我院141例新辅助化疗患者,通过查体、B超和钼靶分别测量记录肿瘤状况,分析治疗前后原发灶、淋巴结、受体状态的变化。结论是化疗前腋窝淋巴结的病理判断,不论是穿刺或前哨活检都非常重要;查体、B超及钼靶对肿瘤的判断都有相当的误差;化疗中可采用病灶穿刺评价NAC疗效,但对结果的判断还需综合分析;需检测治疗前后的受体状态,根据总体分析选择术后靶向ER或HER-2的治疗,争取使患者更多获益。根据此研究结果,我们设计了新辅助的前瞻性临床试验,为下一步基因表达研究提供基础。
第二部分分别研究了冰冻和石蜡包埋(FFPE)的两种乳腺癌组织的RNA提取及基因表达情况。医院病理科有大量的档案FFPE样本,由于乳腺癌患者的生存期较长,可收集保存数年的FFPE样本进行回顾性研究而获得大量的基因信息,用于研究肿瘤的分子分类、分型及对治疗的反应、肿瘤复发转移和预后的潜在生物学标记等方面,具有重要的应用前景。但FFPE样本RNA的提取存在一定困难, RNA降解较为严重。因此我们分别探索从冰冻和石蜡包埋的两种乳腺癌样本中提取总RNA的质量和产量,并检测其基因的表达情况。方法是收集保存时间1~17年不等的人乳腺癌石蜡组织样本153例,收集冰冻及其石蜡包埋的人乳腺癌组织共5对,取5μm厚切片1～15张,试剂盒提取总RNA。随机引物法将mRNA反转录为cDNA,SYBR Green ?染料法实时荧光定量PCR分析两种样本的mRNA表达情况,并对不同保存时间的石蜡样本,比较扩增管家基因不同产物长度的Ct值差异,以了解RNA降解程度。结果,153例石蜡标本总RNA浓度在3～375ng/μl范围,总产量0.18～18.5μg,A260/280值在1.32～2.07范围,RNA片段弥散在100~1000bp之间,峰值在200bp左右,冰冻样本RNA可见清晰的28S、18S条带。石蜡样本扩增90bp长度ACTB基因的有效RNA模板量仅占相应冰冻样本的0.46倍(p<0.05),两种样本的RNA完整性有显著差别。冰冻样本扩增90bp产物的Ct值显著低于203bp的Ct值(p=0.02),提示冰冻样本RNA也有一定降解。石蜡样本mRNA表达与相应的冰冻样本无显著差异(p>0.05),相关性分析显示二者有显著相关性。81例样本的4个管家基因的平均Ct值在24～30范围,随着样本保存时间的延长,Ct值也逐渐增加。扩增不同保存时间石蜡样本的三个长度片段的Ct值均有显著差异,随着扩增片段的增大,Ct值也逐渐增加。结论是冰冻样本的RNA相对较完整,质量较好;石蜡样本的RNA有明显降解,但尚能够准确反应组织mRNA水平上的表达情况;FFPE样本的RNA量可满足实验的要求,可以进行mRNA的检测,保存10年以上的样本也能够进行基因表达研究;石蜡样本的Ct值随扩增片段的增大而逐渐增加,能顺利扩增150bp左右长度的产物,为石蜡样本的进一步广泛研究提供实验基础。
第三部分探讨了乳腺癌患者的21基因表达研究及预后分析。目前关于乳腺癌的预后判断仍停留在传统的基于临床特征的基础上,任何一种方法都不能准确无误地判断患者的预后,因此治疗也就会变得盲目、不足或过度。近年来国外学者进行了一些大样本、高通量的基因表达研究,21基因即是其中最好的研究之一。由21个基因表达水平计算出的复发分数(RS)来判断危险性,对预后有显著预测意义。但RS检测花费昂贵,尚不能在中国进行,且RS没有在中国患者中的数据结果,因此我们试图采用我们的实验方法研究RS在中国乳腺癌患者中的预后价值及应用前景。方法是根据我们制定的患者筛选条件,收集我院病理科的乳腺癌FFPE样本及部分外院样本。试剂盒提取总RNA,采用随机引物将mRNA反转录为cDNA,SYBR Green ?染料法实时荧光定量PCR检测表达情况,据文献报道方法计算RS。结果,共收集石蜡样本109例,有16例样本的RT-PCR扩增结果较差,共有93例有完整的结果。全组为激素受体(HR)阳性、淋巴结(LN)阳性或阴性的早期乳腺癌患者,中位随访时间65.9个月。主要研究终点是无远处复发生存(DRFS),约半数患者(45/93)发生了远处复发。采用文献报道的QRT-PCR判断ER、PR和HER-2状态的界值,将mRNA水平分别与IHC/FISH检测的相应蛋白水平进行比较。这两种方法检测ER、PR和HER-2的Kappa系数分别是0.59、0.44和0.63。RS评估危险度后,低危、中危、高危三组的远处复发率有显著差异(p<0.001),高危组的DRFS显著低于低危和中危组,RS对预后有预测意义。亚组分析中,RS在LN阴性(p=0.009)和阳性者(p=0.038)中均有预后意义。单因素及多因素分析显示LN状态、辅助内分泌治疗和RS均是显著的预后因素。与其他危险评估方法相比,在预后差患者中,RS判断需要给予辅助化疗的比例较St. Gallen共识判断的结果小(81.6% vs 94.7%),但无统计学差异(p>0.05);而在预后较好者中,RS分组认为需给予化疗以降低复发风险的患者显著减低(50.9% vs 90.9%,P<0.001)。结论,我们建立了简便、经济的QRT-PCR方法,其判断ER和HER-2状态与IHC的结果一致性较好,判断PR的结果一般,与原21基因的Taqman探针法结果相似、具有可比性。RS与LN阴性或阳性、HR阳性乳腺癌患者的远处转移有显著的相关性。RS较传统的危险评估方法能更准确地判断预后,并降低辅助化疗的比例,初步探讨了RS在中国乳腺癌患者中的预后价值及临床应用前景。
第四部分研究了乳腺癌患者的microRNA表达情况及临床分析。microRNA(miRNA)在转录后对各种基因进行调控,在细胞增殖、生长、分化、以及肿瘤的发生发展中发挥重要的调节作用。由于miRNA作用机制及调控的复杂多样性,真正确认功能的miRNA较少。目前已有一些实验证实了与乳腺癌侵袭、转移相关的miRNA,并初步在临床样本中得到了数据支持体外实验的结果。我们参考文献报道,选取了几个与乳腺癌转移、临床特征相关的miRNA,初步探讨其在乳腺癌样本中的表达情况以及与临床特征和转归的关系。方法为试剂盒提取包含miRNA的总RNA,采用特异性含茎环结构的反转录引物分别反转录,用Taqman探针法检测miRNA的表达。结果,共有57例样本,其中miR-373的Ct值多>35.0。本组样本的miRNA表达情况服从正态分布,根据ΔCt值的均数分为高表达和低表达两组。主要研究终点是无病生存期(DFS)。仅有miR-21高表达与肿瘤较小有显著关系(p=0.046),miR-206与ER蛋白的表达无显著相关性(p=0.449)。miR-10b对全组患者的预后有预测意义,miR-335、-10b和-21对HR阴性的患者有预后意义,均为高表达者预后较好,而与HR阳性者预后无显著关系。其中,miR-335和-10b与骨转移显著相关(p=0.015)。结论,miR-373在乳腺癌组织中表达很低,结果不一定可靠,难以分析;miR-21高表达与肿瘤较小显著相关;miR-206与ER蛋白的表达无显著相关性;miR-10b高表达者预后较好,HR阴性者中,miR-335、-10b和-21高表达的预后较好;miR-335和-10b高表达发生骨转移显著减少。
总之,本研究从FFPE样本中研究mRNA及miRNA的表达情况,为从乳腺癌样本中开展大量的回顾性或前瞻性研究提供了实验基础。在此基础上,采用简便经济的方法研究了21基因在中国乳腺癌患者的表达情况,验证了RS对预后的预测能力及临床应用前景。从miRNA角度研究了与临床转归相关的重要miRNA,为进一步地研究miRNA功能及其靶基因提供了临床数据资料。对乳腺癌新辅助化疗中不同疗效评价手段及受体变化进行了初步探讨,为今后开展正规、标准的新辅助化疗提供了方向,并设计了新辅助前瞻性临床试验,为进一步基因表达研究提供基础。
The use of archived samples with well-documented clinical characteristics and follow-up accelerates retrospective research and the discovery of potentially useful clinical gene expression signatures. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) specimens is fragmental and poor substrates for cDNA synthesis and subsequent PCR amplification. However, there have been successful researches about the use of RNA isolated from FFPE for real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR) recently. And identification of molecular characteristics that allow an accurate prediction of a patient survival remains an important facet in the current management of cancer.
In the first part of the study, we compared the difference of response evaluation among clinical examination, ultrasonograghy and mammography of neoadjuvant chemotherapy (NAC) in breast cancer. And we analyzed the change of tumor characteristic before and after treatment. A retrospective cohort study was analyzed among 141 patients treated with NAC. Response evaluation was performed by clinical palpation, ultrasound and mammography. Only 12 pts (8.5%) presented with stage ? tumors. Tumor size determined by palpation was often larger than that by ultrasound before therapy (p<0.01). Among patients with axillary nodes checked by ultrasound, 84.6%(55/65) of them had positive nodes by pathology before NAC. And 34.5% (10/29) patients with negative nodes determined by ultrasound had positive nodes by pathology. 56.3% (36/64) of patients with positive nodes by pathology before NAC became pathologic remission after chemotherapy. In all, 14.9% (21/141) pts showed pathology complete regression both in the primary tumor and lymph node. For response evaluation, the false complete remission rate judged by clinical examination was 46.8% (22/47), and the false tumor residual rate by ultrasound was 84.0% (21/25). 53.5% (23/43) pts couldn’t assess response by using mammography due to undistinguishable tumor size. The range of microcalcification didn’t reduce in 5 pts with tumor partly response. 25 pts experienced needle puncture during therapy. Among 9 pts pathology negative, only 3 achieved ppthologic complete remission (pCR), and the other 16 positive patients didn’t achieve pCR. In conclusion, by using the method of puncture or sentinel lymph node biopsy, the clinicians should pay enough emphasis on the pathologic determination of nodes before chemotherapy. Clinicians will make a quite of false judgment of tumor by use clinical examination, ultrasound and mammography. They may use needle puncture during therapy to evaluate response of neoadjuvant chemotherapy, and the result should be analyzed synthetically. On the basement of comparison of the receptor status, the medical can chose HER-2 targeted therapy and endocrine therapy after surgery to benefit patients.
In the second part of the study, we analyzed the RNA extraction and gene expression of frozen and paraffin-embedded breast cancer tissues. We collected 153 cases of paraffin-embedded human breast cancer samples with storage time from 1 to17 years and 5 paired of frozen and FFPE human breast cancer tissues. Total RNA was extracted by using two commercial extraction kit from 1~15 slices of 5μm in thickness. mRNAs were reversely transcripted into cDNA with random primers, and four different housekeeping genes and target genes in mRNA level were detected by SYBR Green ? dye method for real-time fluorescence quantitative PCR reaction analysis. We measured the amplification results of three fragments from ACTB gene to assess the intensity of RNA from FFPE tissues. Of all the 153 samples, total RNA ranged from 3 to 375ng/μl in concentrations, 0.18 ~ 18.5μg in total yield, 1.32 ~ 2.07 in A260/280. Fragments dispersed between 100 ~ 1000bp by RNA agarose gel electrophoresis and Agilent 2100. Compared to frozen samples, an average ~54% loss of intact amplicon template for amplification ACTB (90bp) gene in the corresponding FFPE materials (p>0.05). The integrity of RNA of them had significant differences. The Ct value for amplification of 90bp fragment was lower than that of 203bp fragment (p=0.02), which indicated that RNA from frozen samples was relatively intact. No difference in gene expression was found between frozen and FFPE samples (p>0.05). And a good correlation of them with Spearman correlation coefficient r=0.954. The expression of four housekeeping genes in 81 cases ranged from 24 to 30 in Ct value on average. There had statistical difference between Ct values of three fragments of FFPE tissues. And the Ct value increased with the age of the archival samples from which the RNA was extracted, indicating a high level of RNA degradation in these samples. The result indicated that RNA from frozen samples is relatively intact. RNA from paraffin samples is obvious degraded, but it can accurately reflect the expression of mRNA and could meet the requirements of the experiment. The detection of target gene in mRNA level could be carried out with the degraded total RNA, even in samples stored more than 10 years. Ct value gradually increased with the fragment length. 90~150bp is a favorable length of product for amplification. This research provides an experimental basis for further extensive research of FFPE samples.
In the third part of the study, we investigate gene expression profiling for breast cancer prognosis in Chinese populations. The 21-gene assay is the representative result of combining high-throughput QRT-PCR, optimization of the normalization method, and the development of RS algorithm based on mRNA expression levels. RS was a more accurate predictor of relapse than standard clinical characteristics for patients with hormone receptor (HR) positive, node negative operable breast cancer from original papers. The training set of RS assay was from patients in NSABP B-14 and B-20 trials which didn’t include Chinese patients. And the assay can’t be performed in China and it’s very expensive, we therefore have sought to develop a low-cost approach through some adjustments of experiment processes to assess the predictive value of RS in Chinese. So, the aim of the study is to validate a quantitative RT-PCR assay different from 21-gene assay which can be used to prognosticate the risk of recurrence in patients with ER-positive, lymph node (LN)-negative breast cancer. To accurately determine the relationship between the Recurrence Score (RS) derived from our assay and the risk of distant recurrence in Chinese patients with LN - negative and positive breast cancer through the analysis of paraffin tissues. We obtained archival paraffin-embedded tissues from patients with invasive breast cancer and different axillary lymph node involvement. Quantitative RT-PCR reaction was performed by using the method of SYBR Green ? dye with primers. Expression of the 21 genes was converted to RS by a prespecified algorithm. We then assessed the probability of the test to accurately predict distant recurrence-free survival in this retrospective cohort. Ninety-three patients were eligible based on gene expression profiles. In our population, most breast cancer patients were premenopausal (82.6%), at early stage (93.6%) and ER-positive (91.4%). Median follow-up was 65.9 months. The 5-year relapse-free survival rate for the group was 58.8%. The concordance between the RT-PCR and immunohistochemical (IHC) measurement for ER, PR and HER2 determinations was high and comparable. High RS was predictive of an elevated risk of relapse (P < 0.001). In subgroups of patients, RS had significantly predictive performance both in node-negative (P = 0.009) and node-positive patients (P = 0.038). Multivariable analysis showed that nodal status, adjuvant hormonal therapy and RS were significantly related to prognosis. RS category is a better predictor than the other risk assessment criteria or clinicopatholic features, with which we can determine more accurately the risks for relapse of various patients. We have established an easy and economical quantitative RT-PCR assay and validated in concordance with IHC measurements for ER, PR, and HER-2. RS was associated with distant recurrence among Chinese patients with hormone receptor (HR)-positive breast cancer. This study may promote the use of RS estimated from the expression of the 21 gene set for prognostication and routine clinical diagnostic application in Chinese populations.
In the fourth part of the study, we investigated the expression of microRNA and the association with clinical prognosis in breast cancer patients. miRNAs have been reported to be involved in tumorigenesis and metastasis. So, we selected seven important miRNAs (miR-21, -335, -373, -10b, -206, -210 and let-7a) to examine the expression of them in breast cancer cases by using Taqman RT-PCR. There were 57 breast cancer patients. The distribution of expression levels of miRNAs atΔCt value followed the normal distribution by the test of normality. The cases were divided into two groups according to the each mean level of miRNAs. The primary endpoint was desease-free survival (DFS). The high level expression of miR-21 was significantly correlated with small tumor size (P = 0.046). There had no relationship between the expression of miR-206 and ER protein. The low expression of miR-10b was significantly coreelated with shortened survival of the patients. And in HR-negative patients, the high level of miR-335, -10b and -21 correlated with good clinical prognosis. Overexpression of miR-335 and -10b correlated with less rate of bone metastasis (p=0.015). This study could identify the differentiated miRNAs expression profile in breast cancer and reveal that miR-335, -10b and -21 overexpression was correlated with good breast cancer progonsis, indicating that them may serve as a molecular prognostic marker for breast cancer.
第一部分对乳腺癌的新辅助治疗中临床存在的一些问题进行了初步探讨。乳腺癌新辅助化疗(NAC)目前已成为乳腺癌的标准治疗,治疗后获得病理学完全缓解(pCR)的患者预后最好,因此在我国也普遍开展。但国外关于NAC疗效评价手段的研究不够详细,国内更是鲜有报道,因此我们主要比较了查体、B超和钼靶在乳腺癌NAC疗效评价中的差异,并分析治疗前后肿瘤特征的变化。方法是对我院141例新辅助化疗患者,通过查体、B超和钼靶分别测量记录肿瘤状况,分析治疗前后原发灶、淋巴结、受体状态的变化。结论是化疗前腋窝淋巴结的病理判断,不论是穿刺或前哨活检都非常重要;查体、B超及钼靶对肿瘤的判断都有相当的误差;化疗中可采用病灶穿刺评价NAC疗效,但对结果的判断还需综合分析;需检测治疗前后的受体状态,根据总体分析选择术后靶向ER或HER-2的治疗,争取使患者更多获益。根据此研究结果,我们设计了新辅助的前瞻性临床试验,为下一步基因表达研究提供基础。
第二部分分别研究了冰冻和石蜡包埋(FFPE)的两种乳腺癌组织的RNA提取及基因表达情况。医院病理科有大量的档案FFPE样本,由于乳腺癌患者的生存期较长,可收集保存数年的FFPE样本进行回顾性研究而获得大量的基因信息,用于研究肿瘤的分子分类、分型及对治疗的反应、肿瘤复发转移和预后的潜在生物学标记等方面,具有重要的应用前景。但FFPE样本RNA的提取存在一定困难, RNA降解较为严重。因此我们分别探索从冰冻和石蜡包埋的两种乳腺癌样本中提取总RNA的质量和产量,并检测其基因的表达情况。方法是收集保存时间1~17年不等的人乳腺癌石蜡组织样本153例,收集冰冻及其石蜡包埋的人乳腺癌组织共5对,取5μm厚切片1～15张,试剂盒提取总RNA。随机引物法将mRNA反转录为cDNA,SYBR Green ?染料法实时荧光定量PCR分析两种样本的mRNA表达情况,并对不同保存时间的石蜡样本,比较扩增管家基因不同产物长度的Ct值差异,以了解RNA降解程度。结果,153例石蜡标本总RNA浓度在3～375ng/μl范围,总产量0.18～18.5μg,A260/280值在1.32～2.07范围,RNA片段弥散在100~1000bp之间,峰值在200bp左右,冰冻样本RNA可见清晰的28S、18S条带。石蜡样本扩增90bp长度ACTB基因的有效RNA模板量仅占相应冰冻样本的0.46倍(p<0.05),两种样本的RNA完整性有显著差别。冰冻样本扩增90bp产物的Ct值显著低于203bp的Ct值(p=0.02),提示冰冻样本RNA也有一定降解。石蜡样本mRNA表达与相应的冰冻样本无显著差异(p>0.05),相关性分析显示二者有显著相关性。81例样本的4个管家基因的平均Ct值在24～30范围,随着样本保存时间的延长,Ct值也逐渐增加。扩增不同保存时间石蜡样本的三个长度片段的Ct值均有显著差异,随着扩增片段的增大,Ct值也逐渐增加。结论是冰冻样本的RNA相对较完整,质量较好;石蜡样本的RNA有明显降解,但尚能够准确反应组织mRNA水平上的表达情况;FFPE样本的RNA量可满足实验的要求,可以进行mRNA的检测,保存10年以上的样本也能够进行基因表达研究;石蜡样本的Ct值随扩增片段的增大而逐渐增加,能顺利扩增150bp左右长度的产物,为石蜡样本的进一步广泛研究提供实验基础。
第三部分探讨了乳腺癌患者的21基因表达研究及预后分析。目前关于乳腺癌的预后判断仍停留在传统的基于临床特征的基础上,任何一种方法都不能准确无误地判断患者的预后,因此治疗也就会变得盲目、不足或过度。近年来国外学者进行了一些大样本、高通量的基因表达研究,21基因即是其中最好的研究之一。由21个基因表达水平计算出的复发分数(RS)来判断危险性,对预后有显著预测意义。但RS检测花费昂贵,尚不能在中国进行,且RS没有在中国患者中的数据结果,因此我们试图采用我们的实验方法研究RS在中国乳腺癌患者中的预后价值及应用前景。方法是根据我们制定的患者筛选条件,收集我院病理科的乳腺癌FFPE样本及部分外院样本。试剂盒提取总RNA,采用随机引物将mRNA反转录为cDNA,SYBR Green ?染料法实时荧光定量PCR检测表达情况,据文献报道方法计算RS。结果,共收集石蜡样本109例,有16例样本的RT-PCR扩增结果较差,共有93例有完整的结果。全组为激素受体(HR)阳性、淋巴结(LN)阳性或阴性的早期乳腺癌患者,中位随访时间65.9个月。主要研究终点是无远处复发生存(DRFS),约半数患者(45/93)发生了远处复发。采用文献报道的QRT-PCR判断ER、PR和HER-2状态的界值,将mRNA水平分别与IHC/FISH检测的相应蛋白水平进行比较。这两种方法检测ER、PR和HER-2的Kappa系数分别是0.59、0.44和0.63。RS评估危险度后,低危、中危、高危三组的远处复发率有显著差异(p<0.001),高危组的DRFS显著低于低危和中危组,RS对预后有预测意义。亚组分析中,RS在LN阴性(p=0.009)和阳性者(p=0.038)中均有预后意义。单因素及多因素分析显示LN状态、辅助内分泌治疗和RS均是显著的预后因素。与其他危险评估方法相比,在预后差患者中,RS判断需要给予辅助化疗的比例较St. Gallen共识判断的结果小(81.6% vs 94.7%),但无统计学差异(p>0.05);而在预后较好者中,RS分组认为需给予化疗以降低复发风险的患者显著减低(50.9% vs 90.9%,P<0.001)。结论,我们建立了简便、经济的QRT-PCR方法,其判断ER和HER-2状态与IHC的结果一致性较好,判断PR的结果一般,与原21基因的Taqman探针法结果相似、具有可比性。RS与LN阴性或阳性、HR阳性乳腺癌患者的远处转移有显著的相关性。RS较传统的危险评估方法能更准确地判断预后,并降低辅助化疗的比例,初步探讨了RS在中国乳腺癌患者中的预后价值及临床应用前景。
第四部分研究了乳腺癌患者的microRNA表达情况及临床分析。microRNA(miRNA)在转录后对各种基因进行调控,在细胞增殖、生长、分化、以及肿瘤的发生发展中发挥重要的调节作用。由于miRNA作用机制及调控的复杂多样性,真正确认功能的miRNA较少。目前已有一些实验证实了与乳腺癌侵袭、转移相关的miRNA,并初步在临床样本中得到了数据支持体外实验的结果。我们参考文献报道,选取了几个与乳腺癌转移、临床特征相关的miRNA,初步探讨其在乳腺癌样本中的表达情况以及与临床特征和转归的关系。方法为试剂盒提取包含miRNA的总RNA,采用特异性含茎环结构的反转录引物分别反转录,用Taqman探针法检测miRNA的表达。结果,共有57例样本,其中miR-373的Ct值多>35.0。本组样本的miRNA表达情况服从正态分布,根据ΔCt值的均数分为高表达和低表达两组。主要研究终点是无病生存期(DFS)。仅有miR-21高表达与肿瘤较小有显著关系(p=0.046),miR-206与ER蛋白的表达无显著相关性(p=0.449)。miR-10b对全组患者的预后有预测意义,miR-335、-10b和-21对HR阴性的患者有预后意义,均为高表达者预后较好,而与HR阳性者预后无显著关系。其中,miR-335和-10b与骨转移显著相关(p=0.015)。结论,miR-373在乳腺癌组织中表达很低,结果不一定可靠,难以分析;miR-21高表达与肿瘤较小显著相关;miR-206与ER蛋白的表达无显著相关性;miR-10b高表达者预后较好,HR阴性者中,miR-335、-10b和-21高表达的预后较好;miR-335和-10b高表达发生骨转移显著减少。
总之,本研究从FFPE样本中研究mRNA及miRNA的表达情况,为从乳腺癌样本中开展大量的回顾性或前瞻性研究提供了实验基础。在此基础上,采用简便经济的方法研究了21基因在中国乳腺癌患者的表达情况,验证了RS对预后的预测能力及临床应用前景。从miRNA角度研究了与临床转归相关的重要miRNA,为进一步地研究miRNA功能及其靶基因提供了临床数据资料。对乳腺癌新辅助化疗中不同疗效评价手段及受体变化进行了初步探讨,为今后开展正规、标准的新辅助化疗提供了方向,并设计了新辅助前瞻性临床试验,为进一步基因表达研究提供基础。
The use of archived samples with well-documented clinical characteristics and follow-up accelerates retrospective research and the discovery of potentially useful clinical gene expression signatures. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) specimens is fragmental and poor substrates for cDNA synthesis and subsequent PCR amplification. However, there have been successful researches about the use of RNA isolated from FFPE for real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR) recently. And identification of molecular characteristics that allow an accurate prediction of a patient survival remains an important facet in the current management of cancer.
In the first part of the study, we compared the difference of response evaluation among clinical examination, ultrasonograghy and mammography of neoadjuvant chemotherapy (NAC) in breast cancer. And we analyzed the change of tumor characteristic before and after treatment. A retrospective cohort study was analyzed among 141 patients treated with NAC. Response evaluation was performed by clinical palpation, ultrasound and mammography. Only 12 pts (8.5%) presented with stage ? tumors. Tumor size determined by palpation was often larger than that by ultrasound before therapy (p<0.01). Among patients with axillary nodes checked by ultrasound, 84.6%(55/65) of them had positive nodes by pathology before NAC. And 34.5% (10/29) patients with negative nodes determined by ultrasound had positive nodes by pathology. 56.3% (36/64) of patients with positive nodes by pathology before NAC became pathologic remission after chemotherapy. In all, 14.9% (21/141) pts showed pathology complete regression both in the primary tumor and lymph node. For response evaluation, the false complete remission rate judged by clinical examination was 46.8% (22/47), and the false tumor residual rate by ultrasound was 84.0% (21/25). 53.5% (23/43) pts couldn’t assess response by using mammography due to undistinguishable tumor size. The range of microcalcification didn’t reduce in 5 pts with tumor partly response. 25 pts experienced needle puncture during therapy. Among 9 pts pathology negative, only 3 achieved ppthologic complete remission (pCR), and the other 16 positive patients didn’t achieve pCR. In conclusion, by using the method of puncture or sentinel lymph node biopsy, the clinicians should pay enough emphasis on the pathologic determination of nodes before chemotherapy. Clinicians will make a quite of false judgment of tumor by use clinical examination, ultrasound and mammography. They may use needle puncture during therapy to evaluate response of neoadjuvant chemotherapy, and the result should be analyzed synthetically. On the basement of comparison of the receptor status, the medical can chose HER-2 targeted therapy and endocrine therapy after surgery to benefit patients.
In the second part of the study, we analyzed the RNA extraction and gene expression of frozen and paraffin-embedded breast cancer tissues. We collected 153 cases of paraffin-embedded human breast cancer samples with storage time from 1 to17 years and 5 paired of frozen and FFPE human breast cancer tissues. Total RNA was extracted by using two commercial extraction kit from 1~15 slices of 5μm in thickness. mRNAs were reversely transcripted into cDNA with random primers, and four different housekeeping genes and target genes in mRNA level were detected by SYBR Green ? dye method for real-time fluorescence quantitative PCR reaction analysis. We measured the amplification results of three fragments from ACTB gene to assess the intensity of RNA from FFPE tissues. Of all the 153 samples, total RNA ranged from 3 to 375ng/μl in concentrations, 0.18 ~ 18.5μg in total yield, 1.32 ~ 2.07 in A260/280. Fragments dispersed between 100 ~ 1000bp by RNA agarose gel electrophoresis and Agilent 2100. Compared to frozen samples, an average ~54% loss of intact amplicon template for amplification ACTB (90bp) gene in the corresponding FFPE materials (p>0.05). The integrity of RNA of them had significant differences. The Ct value for amplification of 90bp fragment was lower than that of 203bp fragment (p=0.02), which indicated that RNA from frozen samples was relatively intact. No difference in gene expression was found between frozen and FFPE samples (p>0.05). And a good correlation of them with Spearman correlation coefficient r=0.954. The expression of four housekeeping genes in 81 cases ranged from 24 to 30 in Ct value on average. There had statistical difference between Ct values of three fragments of FFPE tissues. And the Ct value increased with the age of the archival samples from which the RNA was extracted, indicating a high level of RNA degradation in these samples. The result indicated that RNA from frozen samples is relatively intact. RNA from paraffin samples is obvious degraded, but it can accurately reflect the expression of mRNA and could meet the requirements of the experiment. The detection of target gene in mRNA level could be carried out with the degraded total RNA, even in samples stored more than 10 years. Ct value gradually increased with the fragment length. 90~150bp is a favorable length of product for amplification. This research provides an experimental basis for further extensive research of FFPE samples.
In the third part of the study, we investigate gene expression profiling for breast cancer prognosis in Chinese populations. The 21-gene assay is the representative result of combining high-throughput QRT-PCR, optimization of the normalization method, and the development of RS algorithm based on mRNA expression levels. RS was a more accurate predictor of relapse than standard clinical characteristics for patients with hormone receptor (HR) positive, node negative operable breast cancer from original papers. The training set of RS assay was from patients in NSABP B-14 and B-20 trials which didn’t include Chinese patients. And the assay can’t be performed in China and it’s very expensive, we therefore have sought to develop a low-cost approach through some adjustments of experiment processes to assess the predictive value of RS in Chinese. So, the aim of the study is to validate a quantitative RT-PCR assay different from 21-gene assay which can be used to prognosticate the risk of recurrence in patients with ER-positive, lymph node (LN)-negative breast cancer. To accurately determine the relationship between the Recurrence Score (RS) derived from our assay and the risk of distant recurrence in Chinese patients with LN - negative and positive breast cancer through the analysis of paraffin tissues. We obtained archival paraffin-embedded tissues from patients with invasive breast cancer and different axillary lymph node involvement. Quantitative RT-PCR reaction was performed by using the method of SYBR Green ? dye with primers. Expression of the 21 genes was converted to RS by a prespecified algorithm. We then assessed the probability of the test to accurately predict distant recurrence-free survival in this retrospective cohort. Ninety-three patients were eligible based on gene expression profiles. In our population, most breast cancer patients were premenopausal (82.6%), at early stage (93.6%) and ER-positive (91.4%). Median follow-up was 65.9 months. The 5-year relapse-free survival rate for the group was 58.8%. The concordance between the RT-PCR and immunohistochemical (IHC) measurement for ER, PR and HER2 determinations was high and comparable. High RS was predictive of an elevated risk of relapse (P < 0.001). In subgroups of patients, RS had significantly predictive performance both in node-negative (P = 0.009) and node-positive patients (P = 0.038). Multivariable analysis showed that nodal status, adjuvant hormonal therapy and RS were significantly related to prognosis. RS category is a better predictor than the other risk assessment criteria or clinicopatholic features, with which we can determine more accurately the risks for relapse of various patients. We have established an easy and economical quantitative RT-PCR assay and validated in concordance with IHC measurements for ER, PR, and HER-2. RS was associated with distant recurrence among Chinese patients with hormone receptor (HR)-positive breast cancer. This study may promote the use of RS estimated from the expression of the 21 gene set for prognostication and routine clinical diagnostic application in Chinese populations.
In the fourth part of the study, we investigated the expression of microRNA and the association with clinical prognosis in breast cancer patients. miRNAs have been reported to be involved in tumorigenesis and metastasis. So, we selected seven important miRNAs (miR-21, -335, -373, -10b, -206, -210 and let-7a) to examine the expression of them in breast cancer cases by using Taqman RT-PCR. There were 57 breast cancer patients. The distribution of expression levels of miRNAs atΔCt value followed the normal distribution by the test of normality. The cases were divided into two groups according to the each mean level of miRNAs. The primary endpoint was desease-free survival (DFS). The high level expression of miR-21 was significantly correlated with small tumor size (P = 0.046). There had no relationship between the expression of miR-206 and ER protein. The low expression of miR-10b was significantly coreelated with shortened survival of the patients. And in HR-negative patients, the high level of miR-335, -10b and -21 correlated with good clinical prognosis. Overexpression of miR-335 and -10b correlated with less rate of bone metastasis (p=0.015). This study could identify the differentiated miRNAs expression profile in breast cancer and reveal that miR-335, -10b and -21 overexpression was correlated with good breast cancer progonsis, indicating that them may serve as a molecular prognostic marker for breast cancer.
引文
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[30] Bibikova M, Yeakley JM, Chudin E, et al. Gene expression profiles in formalin-fixed , paraffin-embedded tissues obtained with a novel assay for microarray analysis. Clin Chem. 2004, 50(12):2384-6
[31] Bibikova M, Talantov D, Chudin E, et al. Quantitative Gene Expression Profiling in Formalin-Fixed, Paraffin-Embedded Tissues Using Universal Bead Arrays. Am J Pathol. 2004, 165(5):1799-807
[32] Xi YG, Nakajima G, Gavin E, et al. Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA. 2007, 13(10):1668-74
[33] Goldstein LJ, Gray R, Badve S, et al. Prognostic utility of the 21-gene assay in hormone receptor-positive operable breast cancer compared with classical clinicopathologic features. J Clin Oncol. 2008, 26(25):4063-71
[34] Paik S. Methods for Gene Expression Profiling in Clinical Trials of Adjuvant Breast Cancer Therapy. Clin Cancer Res. 2006, 12(3 Suppl):1019s-23s
[35] Cobleigh MA, Tabesh B, Bitterman P, et al. Tumor gene expression and prognosis in breast cancer patients with 10 or nore positive lymph nodes. Clin Cancer Res. 2005, 11(24):8623-31
[36] Chang JC, Makris A, Gutierrez MC, et al. Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients. Breast Cancer Res Treat. 2008, 108(2):233–40
[37] Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol. 2005, 23(29):7265-77
[38] Paik S. Development and Clinical Utility of a 21-Gene Recurrence Score Prognostic Assay in Patients with Early Breast Cancer Treated with Tamoxifen. Oncologist. 2007, 12(6):631-5
[39] Fisher B, Dignam J, Bryant J, et al. Five Versus More Than Five Years of Tamoxifen for Lymph Node-Negative Breast Cancer: Updated Findings From the National Surgical Adjuvant Breast and Bowel Project B-14 Randomized Trial. J Natl Cancer Inst. 2001, 93(9):684-90
[40] Fisher B, Jeong J-H, Bryant J, et al. Treatment of lymph-node-negative, oestrogen-receptor-positive breast cancer: long-term findings from National Surgical Adjuvant Breast and Bowel Project randomised clinical trials. Lancet. 2004, 364(9437):858-68
[41] Esteva F, Sahin AA, Cristofanilli M, et al. Prognostic role of a Multigene Reverse Transcriptase-PCR Assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy. Clin Cancer Res. 2005, 11(9):3315-9
[42] Goldhirsch A, Wo WC, Gelber RD, et al. Progress and promise: highlights of the international expert consensus on the primary therapy of early breast cancer 2007. Anna Oncol. 2007, 18(7):1133–44
[43] Eifel P, Axelson JA, Costa J, et al. National Institutes of Health Consensus Development Conference Statement: Adjuvant Therapy for Breast Cancer, November 1–3, 2000. J Natl Cancer Inst. 2001, 93(13):979-89
[44] Habel LA, Shak S, Jacobs MK, et al. A population-based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients. Breast Cancer Res. 2006, 8(3):R25
[45] Wolf I, Ben-Baruch N, Shapira-Frommer R, et al. Association between standard clinical and pathologic characteristics and the 21-Gene Recurrence Score in breast cancer patients: a population-based study. Cancer. 2008, 112(4):731-6
[46] Carlson RW, Anderson BO, Burstein HJ, et al. NCCN Invasive Breast Cancer Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2007, 5(3):246-312
[47] Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J ClinOncol. 2007, 25(33):5287-312
[48] NCCN Clinical Practice Guidelines in Oncology? Breast Cancer, (Version 2) 2008, http://www.nccn.org (Accessed 1/9/08).
[49] Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research. 2005, 33(20):e179
[50] YAN L-X, HUANG X-F, SHAO Q, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA. 2008, 142348-60
[51] Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005, 65(14):6029-33
[52] Cheng AM, Byrom MW, Shelton J, et al. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis Nucleic Acids Res. 2005, 33(4):1290-7
[53] Foekens JA, Sieuwerts AM, Smid M, et al. Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer. PNAS. 2008, 105(35):13021–6
[54] Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. . Cancer Res. 2004, 64(11):3753-6
[55] Iorio MV, Ferracin M, Liu C-G, et al. MicroRNA Gene Expression Deregulation in Human Breast Cancer. Cancer Res. 2005, 65(16):7065-70
[56] Kuerer H, Newman L, Smith T, et al. Clinical course of breast cancer patients with complete pathological tumor and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy. J Clin Oncol. 1999, 17460-9
[57] Eralp Y, Smith TL, Altundaf K, et al. Eralp Y, Smith TL, Altundag K, et al. Clinical features associated with a favorable outcome following neoadjuvant chemotherapy in women with localized breast cancer aged 35 years or younger. J Cancer Res Clin Oncol. 2009, 135(1):141-8
[58] Fisher B, Bryant J, Wolmark N, et al. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol. 1998, 162672-85
[59] Rastogi P, Anderson SJ, Bear HD, et al. Rastogi P, Anderson SJ, Bear HD, et al. Preoperative Chemotherapy: Updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27. J Clin Oncol. 2008, 26(5):778-85
[60] Carey L, Dees E, Sawyer L, et al. The triple negative paradox: primary tumorchemosensitivity of breast cancer subtypes. Clin Cancer Res. 2007, 13(8):2329-34
[61] Garber J, Richardson A, Harris L, et al. Neoadjuvant cisplatin (CDDP) in “triple-negative”breast cancer (BC)[A]. 29th Annual San Antonio Breast Cancer Symposium. 2006, Abstract 3074
[1] Saghizadeh M, Brown DJ, Tajbakhsh J, et al. Evaluation of techniques using amplified nucleic acid probes for gene expression profiling. Biomolecular Engineering, 2003, 20(3):97-106
[2] Turner N, Tutt A, Ashworth A. Hallmarks of‘BRCAness’in sporadic cancers, Nat Rev Cancer, 2004, 4: 814
[3] Easton DF, Pooley KA, Dunning AM, et al. Genome-wide association study identifies novel breast cancer susceptibility loci. Nature, 447(7148): 1087-1093
[4] Stacey SN, Manolescu A, Sulem P, et al. Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer. Nat Genet. 2007, 39(7):865-9
[5]Thomas RK, Baker AC, Debiasi RM, et al. High-throughput oncogene mutation profiling in human cancer. Nat Genet. 2007, 39(3):347-51.
[6] Gauthier ML, Berman HK, Miller C, et al. Conditional expression of stress proteins in DCIS predicts future tumor formation. SABCS, abst 49
[7] Gauthier ML, Berman HK, Miller C, et al. Abrogated response to cellular stress identifies DCIS associated with subsequent tumor events and defines basal-like breasttumors.Cancer Cell. 2007, 12(5):479-91
[8]Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001, 98:10869-74
[9] Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA. 2003, 100:8418-23
[10]Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 2004; 351:2817-26.
[11] Albain K, Barlow W, Shak S, et al. Prognostic and predictive value of the 21?gene recurrence score assay in postmenopausal, node?positive, ER?positive breast cancer (S8814,INT0100) SABCS
[12] van't Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature, 2002, 415(6871):530-5
[13] Mook S, Schmidt MK, Viale G, et al. Breast cancer patients with 1-3 positive lymph nodes and a low risk 70-gene profile have an excellent survival. SABCS
[14] Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol, 2005,23(29):7265-77
[15] Bonnefoi H, Potti A, Delorenzi M, et al. Validation of gene signatures that predict the response of breast cancer to neoadjuvant chemotherapy: a substudy of the EORTC 10994/BIG 00-01 clinical trial. Lancet Oncol. 2007, 8(12):1071-8.
[16] Jansen MP, Fokens JA, van Staveren IL, et al. Molecular classification of tamoxifen-resistant breast carcinomas by gene expression profiling. J Clin Oncol. 2005;23:732-740.
[17] Kok M, Linn SC, Wessels LF, et al. Robust cross-platform validation of 81-gene tamoxifen resistance signature, the Amsterdam/Rotterdam collaboration (TAMRO). Breast Cancer Res Treat. 2006; 100(suppl 1):S21. Abstract 47.
[18] Ma XJ, Wang Z, Ryan PD, et al. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell. 2004, 5:607-16
[1] Jacobs S, Thompson E R, Nannya Y, et al. Genome-wide, high-resolution detection of copy number, loss of heterozygosity, and genotypes from formalin-fixed, paraffin-embedded tumor tissue using microarrays [J]. Cancer Res, 2007, 67(6):2544-2551.
[2] Penland SK, Keku TO, Torrice C, et al. RNA expression analysis of formalin-fixed paraffin-embedded tumors [J]. Lab Invest, 2007, 87(4):383-391.
[3] Patel V, Hood BL, Molinolo AA, et al. Proteomic analysis of laser-captured paraffin- embedded tissues:a molecular portrait of head and neck cancer progression [J]. Clin Cancer Res, 2008, 14(4):1002-1014.
[4] Hoefig KP, Thorns C, Roehle A, et al. Unlocking pathology archives for microRNA-profiling [J]. Anticancer Res, 2008, 28(1A):119-123.
[5] Olaussen KA, Soria JC, Park YW, et al. Assessing abnormal gene promoter methylation in paraffin-embedded sputum from patients with NSCLC [J]. Eur J Cancer, 2005, 41(14):2112-2119.
[6] Doleshal M, Magotra AA, Choudhury B, et al. Evaluation and validation of totalRNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues [J]. J Mol Diagn, 2008, 10(3):203-211.
[7] Masuda N, Ohnishi T, Kawamoto S, et al. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples [J]. Nucleic Acids Res, 1999, 15;27(22):4436-4443.
[8] Yamaguchi M, Dieffenbach CW, Connolly R, et al. Effect of different laboratory techniques for guanidinium-phenol-chloroform RNA extraction on A260/A280 and on accuracy of mRNA quantitation by reverse transcriptase-PCR [J]. PCR Methods Appl, 1992, 1(4):286-290.
[9] Abrahamsen HN, Steiniche T, Nexo E, et al. Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients [J]. J Mol Diagn, 2003, 5(1):34-41.
[10] Lewis F, Maughan NJ, Smith V, et al. Unlocking the archive-gene expression in paraffin-embedded tissue [J]. J Pathol, 2001, 195(1): 66-71.
[11] Bibikova M, Yeakley JM, Chudin E, et al. Gene expression profiles in formalin-fixed, paraffin-embedded tissues obtained with a novel assay for microarray analysis [J]. Clin Chem, 2004, 50(12):2384-2386.
[2]张杰,邵志敏,沈镇宙.基因芯片与乳腺癌.肿瘤. 2002, 22(4):
[3] Paik S, Shak S, Tang G, et al. A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer. N Engl J Med. 2004, 351(27):2817-26
[4] Albain K, Barlow W, Shak S, et al. Prognostic and predictive value of the 21-gene recurrence score assay in postmenopausal, node-positive, ERpositive breast cancer (S8814,INT0100) Paper presented at 30th Annual San Antonio Breast Cancer Symposium. 2007, San Antonio,TX .Abstract 10
[5] Goldhirsch A, Wood W, Gelber R, et al. Meeting highlights: updated international expert consensus on the primary therapy of early breast cancer. J Clin Oncol. 2003, 21(17):3357-65
[6] van'tVeer LJ, Dai H, vandeVijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002, 415(6871):530-5
[7] Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007, 449682-9
[8] Tavazoie SF, Alarco′n C, Oskarsson T, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature. 2008, 451(10):147-52
[9] Kaufmann M, Minckwitz Gv, Bear HD, et al. Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: new perspectives 2006. Annals of Oncology. 2007, 181927–34
[10] Kaufmann M, Hortobagyi G, A AG, et al. Neoadjuvant Chemotherapy of Breast Cancer. J Clin Oncol. 2006, 241940-9
[11] Bear H, Anderson S, Sminth R, et al. A randomized trial comparing preoperative (preop) doxorubicin/cyclophosphamide(AC) to preop AC followed by postoperative(postop) T in patients(pts) with operable carcinoma of the breast: Result of NSABP B-27. Breast Cancer Res Treat. 2004, 88S16
[12] Bear HD, Anderson S, Brown A, et al. The Effect on Tumor Response of Adding Sequential Preoperative Docetaxel to Preoperative Doxorubicin and Cyclophosphamide: Preliminary Results From National Surgical Adjuvant Breast andBowel Project Protocol B-27 J Clin Oncol. 2003, 21(22):4165-74
[13] Mamounas EP, Brown A, Anderson S, et al. Sentinel Node Biopsy After Neoadjuvant Chemotherapy in Breast Cancer: Results From National Surgical Adjuvant Breast and Bowel Project Protocol B-27. J Clin Oncol. 2005, 232694–702
[14]张斌,张强,赵林, et al.乳腺癌新辅助化疗疗效的评价及影响因素分析.中华肿瘤杂志. 2006, 28(11):867-70
[15] Rupp GM, Locker J. Purification and analysis of RNA from paraffin-embedded tissues. Biotechniques. 1988, 6(1):56-60
[16] Cronin M, Pho M, Dutta D, et al. Measurement of Gene Expression in Archival Paraffin-Embedded Tissues--Development and Performance of a 92-Gene Reverse Transcriptase-Polymerase Chain Reaction Assay. Am J Pathol. 2004, 164(1):35-42
[17] Jacobs S, Thompson ER, Nannya Y, et al. Genome-wide, high-resolution detection of copy number, loss of heterozygosity,and genotypes from formalin-fixed, paraffin-embedded tumor tissue using microarrays. Cancer Res. 2007, 67(6):2544-51
[18] Penland SK, Keku TO, Torrice C, et al. RNA expression analysis of formalin-fixed paraffin-embedded tumors. Lab Invest. 2007, 87(4):383-91
[19] Patel V, Hood BL, Molinolo AA, et al. Proteomic analysis of laser-captured paraffin- embedded tissues:a molecular portrait of head and neck cancer progression. Clin Cancer Res. 2008, 14(4):1002-14
[20] Hoefig KP, Thorns C, Roehle A, et al. ,et al. Unlocking pathology archives for microRNA-profiling. Anticancer Res. 2008, 28(1A):119-23
[21] Olaussen KA, Soria JC, Park YW. Assessing abnormal gene promoter methylation in paraffin-embedded sputum from patients with NSCLC. Eur J Cancer. 2005, 41(14):2112-9
[22] Godfrey TE, Kim S-H, Chavira M, et al. Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 59 Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction. J Mol Diagn. 2000, 2(2):84-91
[23] Ahlfen Sv, Missel A, Bendrat K, et al. Determinants of RNA Quality from FFPE Samples. PLoS ONE. 2007, 2(12):e1261
[24] Masuda N, Ohnishi T, Kawamoto S, et al. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res. 1999, 27(22):4436-43
[25] Gloghini A, Canal B, Klein U, et al. RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues. J Mol Diagn. 2004,6(4):290-6
[26] Doleshal M, Magotra AA, Choudhury B, et al. Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues. J Mol Diagn. 2008, 10(3):203-11
[27] Yamaguchi M, Dieffenbach CW, Connolly R, et al. Effect of different laboratory techniques for guanidinium-phenol-chloroform RNA extraction on A260/A280 and on accuracy of mRNA quantitation by reverse transcriptase-PCR. PCR Methods Appl. 1992, 1(4):286-90
[28] Abrahamsen HN, Steiniche T, Nexo E, et al. Towards Quantitative mRNA Analysis in Paraffin-Embedded Tissues Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction: a Methodological Study on Lymph Nodes from Melanoma Patients. J Mol Diagn. 2003, 5(1):34-41
[29] Lewis F, Maughan NJ, Smith V. Unlocking the archive - gene expression in paraffin-embedded tissue. J Pathol. 2001, 195(1):66-71
[30] Bibikova M, Yeakley JM, Chudin E, et al. Gene expression profiles in formalin-fixed , paraffin-embedded tissues obtained with a novel assay for microarray analysis. Clin Chem. 2004, 50(12):2384-6
[31] Bibikova M, Talantov D, Chudin E, et al. Quantitative Gene Expression Profiling in Formalin-Fixed, Paraffin-Embedded Tissues Using Universal Bead Arrays. Am J Pathol. 2004, 165(5):1799-807
[32] Xi YG, Nakajima G, Gavin E, et al. Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA. 2007, 13(10):1668-74
[33] Goldstein LJ, Gray R, Badve S, et al. Prognostic utility of the 21-gene assay in hormone receptor-positive operable breast cancer compared with classical clinicopathologic features. J Clin Oncol. 2008, 26(25):4063-71
[34] Paik S. Methods for Gene Expression Profiling in Clinical Trials of Adjuvant Breast Cancer Therapy. Clin Cancer Res. 2006, 12(3 Suppl):1019s-23s
[35] Cobleigh MA, Tabesh B, Bitterman P, et al. Tumor gene expression and prognosis in breast cancer patients with 10 or nore positive lymph nodes. Clin Cancer Res. 2005, 11(24):8623-31
[36] Chang JC, Makris A, Gutierrez MC, et al. Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients. Breast Cancer Res Treat. 2008, 108(2):233–40
[37] Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol. 2005, 23(29):7265-77
[38] Paik S. Development and Clinical Utility of a 21-Gene Recurrence Score Prognostic Assay in Patients with Early Breast Cancer Treated with Tamoxifen. Oncologist. 2007, 12(6):631-5
[39] Fisher B, Dignam J, Bryant J, et al. Five Versus More Than Five Years of Tamoxifen for Lymph Node-Negative Breast Cancer: Updated Findings From the National Surgical Adjuvant Breast and Bowel Project B-14 Randomized Trial. J Natl Cancer Inst. 2001, 93(9):684-90
[40] Fisher B, Jeong J-H, Bryant J, et al. Treatment of lymph-node-negative, oestrogen-receptor-positive breast cancer: long-term findings from National Surgical Adjuvant Breast and Bowel Project randomised clinical trials. Lancet. 2004, 364(9437):858-68
[41] Esteva F, Sahin AA, Cristofanilli M, et al. Prognostic role of a Multigene Reverse Transcriptase-PCR Assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy. Clin Cancer Res. 2005, 11(9):3315-9
[42] Goldhirsch A, Wo WC, Gelber RD, et al. Progress and promise: highlights of the international expert consensus on the primary therapy of early breast cancer 2007. Anna Oncol. 2007, 18(7):1133–44
[43] Eifel P, Axelson JA, Costa J, et al. National Institutes of Health Consensus Development Conference Statement: Adjuvant Therapy for Breast Cancer, November 1–3, 2000. J Natl Cancer Inst. 2001, 93(13):979-89
[44] Habel LA, Shak S, Jacobs MK, et al. A population-based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients. Breast Cancer Res. 2006, 8(3):R25
[45] Wolf I, Ben-Baruch N, Shapira-Frommer R, et al. Association between standard clinical and pathologic characteristics and the 21-Gene Recurrence Score in breast cancer patients: a population-based study. Cancer. 2008, 112(4):731-6
[46] Carlson RW, Anderson BO, Burstein HJ, et al. NCCN Invasive Breast Cancer Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2007, 5(3):246-312
[47] Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J ClinOncol. 2007, 25(33):5287-312
[48] NCCN Clinical Practice Guidelines in Oncology? Breast Cancer, (Version 2) 2008, http://www.nccn.org (Accessed 1/9/08).
[49] Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem–loop RT–PCR. Nucleic Acids Research. 2005, 33(20):e179
[50] YAN L-X, HUANG X-F, SHAO Q, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA. 2008, 142348-60
[51] Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005, 65(14):6029-33
[52] Cheng AM, Byrom MW, Shelton J, et al. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis Nucleic Acids Res. 2005, 33(4):1290-7
[53] Foekens JA, Sieuwerts AM, Smid M, et al. Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer. PNAS. 2008, 105(35):13021–6
[54] Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. . Cancer Res. 2004, 64(11):3753-6
[55] Iorio MV, Ferracin M, Liu C-G, et al. MicroRNA Gene Expression Deregulation in Human Breast Cancer. Cancer Res. 2005, 65(16):7065-70
[56] Kuerer H, Newman L, Smith T, et al. Clinical course of breast cancer patients with complete pathological tumor and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy. J Clin Oncol. 1999, 17460-9
[57] Eralp Y, Smith TL, Altundaf K, et al. Eralp Y, Smith TL, Altundag K, et al. Clinical features associated with a favorable outcome following neoadjuvant chemotherapy in women with localized breast cancer aged 35 years or younger. J Cancer Res Clin Oncol. 2009, 135(1):141-8
[58] Fisher B, Bryant J, Wolmark N, et al. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol. 1998, 162672-85
[59] Rastogi P, Anderson SJ, Bear HD, et al. Rastogi P, Anderson SJ, Bear HD, et al. Preoperative Chemotherapy: Updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27. J Clin Oncol. 2008, 26(5):778-85
[60] Carey L, Dees E, Sawyer L, et al. The triple negative paradox: primary tumorchemosensitivity of breast cancer subtypes. Clin Cancer Res. 2007, 13(8):2329-34
[61] Garber J, Richardson A, Harris L, et al. Neoadjuvant cisplatin (CDDP) in “triple-negative”breast cancer (BC)[A]. 29th Annual San Antonio Breast Cancer Symposium. 2006, Abstract 3074
[1] Saghizadeh M, Brown DJ, Tajbakhsh J, et al. Evaluation of techniques using amplified nucleic acid probes for gene expression profiling. Biomolecular Engineering, 2003, 20(3):97-106
[2] Turner N, Tutt A, Ashworth A. Hallmarks of‘BRCAness’in sporadic cancers, Nat Rev Cancer, 2004, 4: 814
[3] Easton DF, Pooley KA, Dunning AM, et al. Genome-wide association study identifies novel breast cancer susceptibility loci. Nature, 447(7148): 1087-1093
[4] Stacey SN, Manolescu A, Sulem P, et al. Common variants on chromosomes 2q35 and 16q12 confer susceptibility to estrogen receptor-positive breast cancer. Nat Genet. 2007, 39(7):865-9
[5]Thomas RK, Baker AC, Debiasi RM, et al. High-throughput oncogene mutation profiling in human cancer. Nat Genet. 2007, 39(3):347-51.
[6] Gauthier ML, Berman HK, Miller C, et al. Conditional expression of stress proteins in DCIS predicts future tumor formation. SABCS, abst 49
[7] Gauthier ML, Berman HK, Miller C, et al. Abrogated response to cellular stress identifies DCIS associated with subsequent tumor events and defines basal-like breasttumors.Cancer Cell. 2007, 12(5):479-91
[8]Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001, 98:10869-74
[9] Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA. 2003, 100:8418-23
[10]Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 2004; 351:2817-26.
[11] Albain K, Barlow W, Shak S, et al. Prognostic and predictive value of the 21?gene recurrence score assay in postmenopausal, node?positive, ER?positive breast cancer (S8814,INT0100) SABCS
[12] van't Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature, 2002, 415(6871):530-5
[13] Mook S, Schmidt MK, Viale G, et al. Breast cancer patients with 1-3 positive lymph nodes and a low risk 70-gene profile have an excellent survival. SABCS
[14] Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol, 2005,23(29):7265-77
[15] Bonnefoi H, Potti A, Delorenzi M, et al. Validation of gene signatures that predict the response of breast cancer to neoadjuvant chemotherapy: a substudy of the EORTC 10994/BIG 00-01 clinical trial. Lancet Oncol. 2007, 8(12):1071-8.
[16] Jansen MP, Fokens JA, van Staveren IL, et al. Molecular classification of tamoxifen-resistant breast carcinomas by gene expression profiling. J Clin Oncol. 2005;23:732-740.
[17] Kok M, Linn SC, Wessels LF, et al. Robust cross-platform validation of 81-gene tamoxifen resistance signature, the Amsterdam/Rotterdam collaboration (TAMRO). Breast Cancer Res Treat. 2006; 100(suppl 1):S21. Abstract 47.
[18] Ma XJ, Wang Z, Ryan PD, et al. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell. 2004, 5:607-16
[1] Jacobs S, Thompson E R, Nannya Y, et al. Genome-wide, high-resolution detection of copy number, loss of heterozygosity, and genotypes from formalin-fixed, paraffin-embedded tumor tissue using microarrays [J]. Cancer Res, 2007, 67(6):2544-2551.
[2] Penland SK, Keku TO, Torrice C, et al. RNA expression analysis of formalin-fixed paraffin-embedded tumors [J]. Lab Invest, 2007, 87(4):383-391.
[3] Patel V, Hood BL, Molinolo AA, et al. Proteomic analysis of laser-captured paraffin- embedded tissues:a molecular portrait of head and neck cancer progression [J]. Clin Cancer Res, 2008, 14(4):1002-1014.
[4] Hoefig KP, Thorns C, Roehle A, et al. Unlocking pathology archives for microRNA-profiling [J]. Anticancer Res, 2008, 28(1A):119-123.
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