乳腺癌免疫组化检测在乳腺癌患者诊治中的意义分析

目的：探讨免疫组化检测在乳腺癌患者诊治中的价值。方法：随机选取2011年1月-2013年1月的68例经过空心穿刺活捡并病理确诊的乳腺癌患者为研究对象,均采用免疫组化检测ER、PR、P53、Bcl-2,全部采用CEF化疗方案治疗3个月后手术治疗,再运用免疫组化SP法检测化疗前后乳腺癌组织中以上指标的阳性表达率情况。结果：ER、PR化疗前后比较无统计学意义（P〉0．05）;而P53、Bcl-2比较有明显的差异性（P〈0．05）;ER、PR的阴性和阳性和疗效情况无明显差异性,而P53、Bcl-2的阴性和阳性表达和化疗的效果有明显的差异性,P〈0．05,具有统计学意义。结论：免疫组化检测中ER、PR对乳腺癌化疗前后无明显差异性,而化疗可通过抑制P53的表达来抑制乳腺癌增值并通过升高Bcl-2表达来调整肿瘤细胞分化。     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

Usefulness of estrogen receptor detection using archival Papanicolaou-stained smears.

OBJECTIVE: To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). STUDY DESIGN: Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. RESULTS: In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. CONCLUSION: Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.     

Estrogen receptor determination in fine needle aspirates of the breast. Correlation with histologic grade and comparison with biochemical analysis

Material obtained by fine needle aspiration (FNA) of 25 surgically removed breast carcinomas was tested for the immunocytochemical localization of estrogen receptor (ER) using the peroxidase-antiperoxidase method and a monoclonal antibody developed against human breast cancer ER. The results were compared to those obtained by the conventional biochemical analysis of cytosol protein. A semiquantitative relationship between the immunoperoxidase stain and the biochemical analysis suggests that cases in which greater than 70% of the cells stain and in which intense staining is present are likely to contain ER in a concentration of greater than 250 fmol/mg of cytosol. Less than 15% stained cells and an absence of intense staining is indicative of a concentration of less than 10 fmol/mg. In only one case was there a significant difference in positivity between the two methods, possibly as a result of a functional heterogeneity of the tumor cell population. Intense staining is strongly suggestive of a tumor of low histologic grade and was never seen in tumors with a high histologic grade or nuclear grade. The immunoperoxidase method of ER detection on material obtained by FNA is a semiquantitative means of selecting patients with breast cancer who are likely to respond to hormonal therapy. The method overcomes many important disadvantages of cytosol analysis and provides clinically significant information regarding the ER content and the degree of tumor differentiation.     

Comparison of two methods of storing breast fine needle aspirates (FNAs) using oestrogen receptor immunocytochemical assay as a method of evaluating the storage methods

Two methods of storing fine needle aspirates were compared in 14 patients with breast cancer. the methods of storage were: (1) as a Cytospin slide prepared immediately from the aspirated material and stored at −80°C; (2) as a suspension of cells in tissue culture medium, stored at −80°C. the effect of storage on the cells was assessed by means of an oestrogen receptor immunocytochemical assay (ER-ICA). an ER positivity of 100% was obtained by ER-ICA staining of cells after storage method 1, whilst all of the specimens stored by method 2 were ER-negative. the data demonstrate that cells stored in tissue culture medium at −80°C are not suitable for ER measurement. the storage method of choice for specimens intended for ERICA is as a Cytospin slide. the ER status of cells deposited on Cytospin slides prepared immediately and stored at −80°C for 2 years could be demonstrated despite the delay in processing the specimen.     

Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation

Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents.     

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.     

Quantitative immunoprofiles of breast cancer performed by image analysis

OBJECTIVE: To determine the biopathologic profiles of breast cancer for greater knowledge of tumor natural history and clinical outcome. STUDY DESIGN: In 99 in situ (ISC) and 2718 infiltrating breast carcinomas (IC), biologic markers (estrogen receptor [ER], progesterone receptor [PR] proliferation index, cerbB-2/NEU, p53, bcl-2 and DNA ploidy) were evaluated with an image analysis system (CAS 200/486). In 105 mixed invasive cancers with size < or = 1 cm, a separate analysis of in situ (ISCm) and invasive component (ICm) was obtained. A clinical study of 836 invasive breast cancers was performed. RESULTS: Different biophenotypes were obtained: among ISCs, cribriform type exhibited biologic behavior similar to that of normal breast tissue (ER+, PR+, proliferation index [PI] low, NEU-, p53-, bcl-2+) the opposite profile was displayed by comedo type, and intermediate phenotypes were observed in noncomedo and lobular types. Comparing ISC and ISCm, PI and p53 expression had the highest levels in ISCm with respect to other groups. NEU overexpression exhibited a decreasing value from ICm to IC. Younger women (< or = 40 years) with IC demonstrated a worse biologic profile (high PI, p53+, ER- and size > 2 cm). In multivariate analysis, PI and NEU in node-negative patients, and NEU, PR and size in node-positive ones emerged as prognostic parameters. CONCLUSION: The results underline the importance of the quantitative biologic profile for defining tumor behavior and patient management.     

Application of multiplex PCR with histopathologic features for detection of familial breast cancer in formalin-fixed, paraffin-embedded histologic specimens

Breast cancer is the most common malignancy among females in the world. Age and familial history are the major risk factors for the development of this disease in Iran. Mutations of BRCA1 and BRCA2 genes are associated with a greatly increased risk for development of familial breast cancer. Frequency of BRCA mutations was identified in familial breast cancers (FBC) and non-familial breast cancers (NFBC) by molecular genetics, morphological and Immunohistochemical methods. Thirty forth formalin-fixed, paraffin-embedded breast tissue tumors were analyzed from 16 patients with FBC and 18 patients with NFBC. Three 5382insC mutations detected by multiplex PCR in 16 familial breast cancers. Immunohistochemical method was used to detect estrogen receptor (ER) and progesterona receptor (PR) and TP53. Comparison of ER, PR and TP53 exhibited high difference (P < 0.0001) in familial breast cancers and non-familial breast cancers. Our results demonstrated that 5382insC mutation, ER, PR, TP53, mitotic activity, polymorphism, necrosis and tubules can serve as the major risk factors for the development of FBC.     

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.     

Immunohistochemical demonstration of estrogen receptors (ER) in formalin-fixed, paraffin-embedded human breast cancer tissue by use of a monoclonal antibody to ER

We describe an immunohistochemical method using a monoclonal antibody to localize estrogen receptors (ER) in formalin-fixed, paraffin-embedded tissue. The avidin-biotin-peroxidase complex method was used, preceded by trypsin treatment to expose antigenic sites. In 111 breast cancer specimens studied simultaneously by a dextran-coated charcoal (DCC) assay and the paraffin section method, agreement on receptor status was found in 101 (91%) specimens. Quantitative staining features showed a high degree of correlation with the results of the steroid binding assay (r = 0.81). Studies on the influence of fixation on ER localization done in rabbit uteri showed that fixatives mainly composed of coagulating reagents (Carnoy's, Zenker's, Bouin's, Lilly's AAF, Helly's, ethanol) precluded ER staining, whereas cross-linking fixatives (formaldehyde, glutaraldehyde) preserved antigenic sites, although the immunoreactivity of the receptor was somewhat decreased. Studies on the effect of enzyme preincubation showed this to increase antigenic expression of ER in formaldehyde-fixed breast tumors and in formaldehyde-, glutaraldehyde-, and Zamboni-fixed rabbit uteri.     

Centchroman induces G0/G1 arrest and caspase-dependent apoptosis involving mitochondrial membrane depolarization in MCF-7 and MDA MB-231 human breast cancer cells

Studies with Centchroman (CC) as a candidate anti-breast cancer agent are into phase III multicentric clinical trial in stage III/IV breast cancer. We have previously demonstrated its anti-neoplastic activity in Estrogen Receptor positive (ER+ve) MCF-7 Human Breast Cancer Cells (HBCCs). We now present the basis for anti-neoplastic activity of CC, mediated through apoptosis in both ER+ve/-ve MCF-7 and MDA MB-231 HBCCs respectively, compared to Tamoxifen (TAM) as a positive control. All the experiments were performed with 48 h estrogen-deprived cells exposed to CC/TAM for the subsequent 48 h. Cytotoxic potential of CC was assessed through SRB assay. Cell-cycle analysis, Time-dependent cytotoxicity, Reactive Oxygen Species (ROS) and Mitochondrial Membrane Permeability were investigated by Flow Cytometry. Early-stage apoptosis was detected by Annexin-PI staining. Caspases were assayed colorimetrically whereas nuclear derangements were assessed morphologically through PI staining and finally by DNA fragmentation analysis. Cell viability studies confirmed the IC50 of CC in MCF-7 and MDA MB-231 cells to be 10 and 20 microM (P < 0.001) respectively, suggesting enhanced susceptibility of the former cell type to CC. FACS data reveals CC mediated G0/G1 arrest (P < 0.01) along with the presence of prominent sub-G0/G1 peak (P < 0.001) in both the cell types suggesting ongoing apoptosis. Phosphatidylserine externalization, mitochondrial events, caspase evaluation and nuclear morphology changes reveal initiation/progression of caspase-dependent apoptosis even at a dose of 1 microM which eventually leads to DNA fragmentation in both the cell types. Results demonstrate that CC induces caspase-dependent apoptosis in MCF-7 and MDA MB-231 cells irrespective of ER status similar to TAM in terms of anti-neoplastic activity.     

三种固定液对两种氧化应激细胞模型Bclin1和LC3蛋白免疫荧光染色的影响

摘要 目的：比较采用三种不同的固定液对两种氧化应激细胞模型Beclin1和LC3蛋白免疫荧光染色的影响。方法：本研究使用丙酮/甲醇（1:1）固定液、甲醇固定液和4%多聚甲醛三种固定液分别对氧化应激细胞模型大鼠原代心肌成纤维细胞和MCF-7乳腺癌细胞株进行固定,然后再分别进行免疫荧光双染实验,对比三种固定液固定后对自噬关键调控蛋白Beclin1和LC3染色效果。结果：三种固定液对氧化应激细胞模型Beclin1和LC3蛋白免疫荧光染色结果存在较大差异。丙酮/甲醇（1:1）固定液固定后免疫荧光染色效果最佳,细胞结构清晰可见,两种蛋白定位表达清晰,甲醇固定液次之,4%多聚甲醛固定液效果欠佳。结论：在对大鼠原代心肌成纤维细胞和MCF-7乳腺癌细胞进行自噬相关蛋白免疫荧光双染色实验中,在使用其它固定液染色效果不佳的情况下,可以选择应用丙酮/甲醇（1:1）固定液固定,再进行免疫荧光染色;根据不同实验需求相应选择更适宜的固定液,以达到最佳的荧光染色结果。     

Estrogen receptor β agonists affect growth and gene expression of human breast cancer cell lines

Expression of estrogen receptor β (ERβ) has been described to reduce growth of cancer cell lines derived from hormone-dependent tumors, like breast cancer. In this study we tested to what extent two ERβ agonists, androgen derivative 3β-Adiol and flavonoid Liquiritigenin, would affect growth and gene expression of different ERβ-positive human breast cancer cell lines. Under standard cell culture conditions, we observed 3β-Adiol to inhibit growth of MCF-7 cells in a dose-dependent manner, whereas growth of BT-474 and MCF-10A cells was suppressed by the maximum concentration (100 nM) only. When treated in serum-free medium, all cell lines except of MDA-MB-231 were responsive to 1 nM 3β-Adiol, and ZR75-1 cells exhibited a dose-dependent antiproliferative response. Providing putative mechanisms underlying the observed growth-inhibitory effect, expression of Ki-67 or cyclins A2 and B1 was downregulated after 3β-Adiol treatment in all responsive lines. In contrast, treatment with lower doses of Liquiritigenin did not affect growth. In MCF-7 cells, the highest dose of this flavonoid exerted proliferative effects accompanied by increased expression of cyclin B1, PR and PS2, indicating unspecific activation of ERα. In conclusion, the ERβ agonists tested exerted distinct concentration-dependent and cell line-specific effects on growth and gene expression. The observed inhibitory effects of 3β-Adiol on breast cancer cell growth encourage further studies on the potential of this and other ERβ agonists as targeted drugs for breast cancer therapy.     

The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations

The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations
We have assessed multiple biological variables on breast carcinoma FNA specimens using a Cytoblock technique. The growth fraction (MIBI), oestrogen receptor (ER), progesterone receptor (PR), p53 mutant protein, c-erbB-2, epidermal growth factor receptor (EGFR), NCRCl Vepithelial membrane antigen (EMA) and DNA plopidy were examined. Objective quantification using image analysis (CAS 200) was applied as appropriate. Fifty cases were examined in this preliminary study. Excellent correlation between the Cytoblock preparations and parallel tissue sections was seen. Of the cancers, 81% were aneuploid with only 19% diploid in character, but 67% of the carcinomas were of histological grade 3. The mean nuclear area staining with MIBl was 31.3% and with ER was 26.7%. Twenty-four percent (24.1%) of the nuclear area showed immunoreactivity with PR. Significant immunostaining was seen in 38%, 46%, 38% and 95% of carcinomas with c-erbB-2, p53, EGFR and EMA, respectively. A significant association between histological grade of the resected tumours and both MIBl (P=0.04) and EGFR (P=0.02) expression in the Cytoblock samples was seen. p53 (P = 0.03) and EGFR (P=0.01) immunoreactivity showed an association with tumour size. EGFR (P=0.04) immunostaining also showed a relationship with the lymph node status of the patient. The technique is, we believe, a useful one for the assessment of multiple variables on breast cytology specimens; these preliminary data suggest that some of these may be useful in predicting prognosis in breast cancer patients.     

Immunocytochemical detection of estrogen receptors by staining with monoclonal antibodies on cytologic specimens of human breast cancer

A study was undertaken to test the possibility of determining the estrogen receptor (ER) content in human breast cancers by staining with commercial specific monoclonal antibodies (MAbs) on cytologic specimens (touch imprints and fine needle aspirates). The aspirates were suspended in a cell culture medium and cytocentrifuged onto slides to preserve their morphologic characteristics and to allow a proper immunocytochemical staining for ERs. MAb staining for ER was also performed on the respective surgical samples. The staining of cytologic samples for ER showed 100% specificity and 95% sensitivity in comparison to the staining of the histologic samples. Moreover, comparison of the percentage of stained cells in the cytologic specimens to the ER content in the respective surgical specimens, as assayed by the dextran-coated charcoal method, showed the MAb staining of cytologic samples to have 94% specificity and 100% sensitivity. These results support the reliability of MAb staining for ERs in cytologic samples and suggest that it could be the assay of choice in particular clinical settings in the evaluation of primary and recurrent breast cancers.     

Immunochemistry for oestrogen receptor,progesterone receptor and HER2 on cell blocks in primary breast carcinoma

K. Kumar S, N. Gupta, A. Rajwanshi, K. Joshi and G. Singh Immunochemistry for oestrogen receptor, progesterone receptor and HER2 on cell blocks in primary breast carcinoma Objective: Steroid receptors and human epidermal growth receptor 2 (HER2) have been used for predicting response to treatment in breast cancers. Fine needle aspiration cytology can provide highly cellular material and can be used for such analysis. The present study was undertaken to assess the reliability of oestrogen and progesterone receptor (ER, PR) status and HER2 as demonstrated by immunochemistry (IHC) on cell blocks from breast carcinoma cases, in comparison with histological sections. Methods: IHC for ER, PR and HER2 was performed on cell blocks and their corresponding tissue sections of 50 primary pre‐chemotherapy breast carcinomas. Positivity for ER and PR was scored according to the Allred scoring system. Strong membranous positivity in more than 30% of tumour cells was considered positive for HER2. The tumours were classified as luminal A, luminal B, HER2‐over‐expressing and triple negative on the basis of ER, PR and HER2 status and results on cell blocks compared with histological sections. Results: Correlation between immunostaining on cell blocks and the corresponding tumour tissues revealed a concordance rate for ER, PR and HER2 of 90% [Correlation coefficient (r) = 0.79], 94% (r = 0.86) and 90% (r = 0.76), respectively. Including five cases in which cell blocks were either ER or PR positive, 43/50 cases (86.0%) could be correctly classified on cell block immunostaining alone. The main reasons for seven discordant cases included technical errors (sampling error and staining error) and interpretational error in HER2 evaluation on cell blocks; the core biopsy was inadequate in one, and apparently false negative for HER2 in another. Conclusion: Cell blocks are useful in the assessment of hormone receptor status and HER2 by IHC, especially in cases of locally advanced breast cancer for planning neoadjuvant chemotherapy. It is highly recommended to have good quality cell blocks and quality control of their interpretation.