Breast Cancer To Neoadjuvant Chemotherapy Judgment And Gene Expression And Prognostic Analysis

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.