Assessment of hormone receptors in breast carcinoma by immunocytochemistry and image analysis. II. Estrogen receptors

Frozen sections of 30 invasive breast carcinomas were stained for estrogen receptors (ERs) and the tumor cell proliferative rate by an immunoalkaline phosphatase technique. The stained sections were evaluated for ER by the microTICAS image analysis system. Seventeen tumors were ER positive and 13 were ER negative by image analysis. There was 93% concordance between the ER results obtained by image analysis and those obtained by biochemical methods. One case that was ER negative by image analysis was weakly positive by biochemical assay; a second case was ER positive by image analysis but ER negative by biochemical assay. Twelve of the 17 ER-positive tumors were diffusely positive while 5 displayed considerable intratumoral heterogeneity, with tumor cells exhibiting a broad range of intensity of receptor expression. In most cases, the image analysis ER status coincided with the progesterone receptor (PR) status, but in a large minority of cases (41%) the ER status and the PR status differed. Tumors with a high growth fraction (greater than 30%), as measured by Ki-67 immunostaining, were uniformly ER negative. The results of this investigation suggest that immunohistochemical staining of frozen sections for ER aided by automated image analysis (1) reliably detects the receptor in breast carcinoma, (2) allows for the assessment of heterogeneity within tumors and (3) may be used as part of a panel of antibodies to markers of potential prognostic importance in a single small tissue sample.     

Quantitative image analysis of estrogen and progesterone receptors as a prognostic tool for selecting breast cancer patients for therapy

OBJECTIVE: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis. STUDY DESIGN: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system. RESULTS: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse. CONCLUSION: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.     

Comparison of different antibodies for detection of progesterone receptor in breast cancer

Monoclonal antibodies directed against human estrogen receptor (ER) and progesterone receptor (PR) have been used extensively for biochemical and immunohistochemical detection of receptors independent of hormone-binding assays. These antibodies have been valuable both for experimental work and for detection of receptors in clinical breast cancer specimens. The purpose of this study was to characterize the sensitivity and specificity of different antibodies for detection of PR by immunohistochemistry (IHC) of formalin-fixed paraffin breast carcinoma sections. The panel of twelve antibodies included two new ones (PgR636 and PgR1294) produced prospectively to be resistant to formalin fixation and paraffin embedding. Fifty-nine breast carcinomas, having known PR levels by biochemical ligand-binding assay, were used to prepare multitumor paraffin-embedded tissue blocks for characterization of the PR antibodies. Of all the antibodies tested, both PgR636 and PgR1294 stained the highest percentage of breast carcinomas known to be positive by the biochemical assay (95-98%) and they exhibited the highest concordance with the biochemical assay (88-90%). The PgR636 and PgR1294 antibodies, along with one other, PR 88, also gave the highest intensity of nuclear staining, while PgR636 and PgR1294 stained the highest mean percentage of tumor cell nuclei. Antigen retrieval was not necessary for PR immunostaining by PgR636 and PgR1294 in most tumors and other tissues examined, but did slightly increase the staining intensity. The majority of the other antibodies tested were highly dependent on antigen retrieval; only PR 88 and KD 68 antibodies approached the performance of PgR636 and PgR1294 without antigen retrieval. These results indicate that PgR636 and PgR1294 are optimal antibodies for IHC detection of PR in routine paraffin tissue blocks.     

Determination of estrogen receptors in cytologic imprints of breast cancers using immunocytochemical methods.

Immunostaining of estrogen receptors (ERs) was carried out on imprints of 62 breast carcinomas using monoclonal antibodies and a sensitive immunoperoxidase technique (the Abbott ER-ICA kit). The results were compared to those obtained by the conventional biochemical analysis of cytosol proteins and to the degree of tumor differentiation. The cytologic specimens were insufficient for analysis in 6 cases; of the remaining 56 cases, 37 (66%) showed a positive ER reaction. In 51 cases with both types of ER analysis, the immunocytochemical staining of the imprints correlated strongly with the biochemical analysis in 44 cases and weakly in 3. Four cases were negative immunocytochemically and positive biochemically. Among the ductal carcinomas, well-differentiated tumors had higher percentages of ER-positive cells than did poorly differentiated tumors. These results show that the immunoperoxidase method is a highly specific and sensitive technique for the evaluation of ER content; it may be applicable to small samples of tumor tissue and may provide additional information for identifying hormonally responsive breast tumors.     

Comparison of estrogen receptor immunocytochemical assay in frozen and paraffin sections.

Frozen and paraffin sections of 11 breast carcinomas were stained for estrogen receptors (ER) using the same rat monoclonal primary antibody, D75P3, as the marker and alkaline phosphatase as the chromogen-linking enzyme. The results of this staining process were assessed visually and with the microTICAS image analysis system to determine the degree of correlation between frozen and paraffin-embedded tissue. In all specimens, some fraction of the nuclei stained positively. This included two specimens selected for their biochemically negative assay; one of them stained strongly positively with D75P3. The results of quantitative analysis support the visually apparent correlation between the two types of samples in terms of both overall staining pattern and intensity of nuclear staining. Although the conclusions of this pilot study are limited because of the small number of cases, this method of staining establishes the feasibility of representative ER determination in archival paraffin-processed material. The additional information provided by this method is potentially useful in stratifying patients in prospective studies on the basis of the efficacy of hormonal therapy in biochemically ER positive breast tumors.     

Relationship between estrogen receptors, 17 beta-hydroxysteroid dehydrogenase and estrogen content in human breast cancer.

Estrone and estradiol levels in tumor tissue cytosols were determined in 11 premenopausal and 20 postmenopausal women at the same time that 17 beta-hydroxysteroid dehydrogenase and estrogen receptors (ER) were carried out on their breast cancers. Estrogen receptor positive tumors showed significantly higher levels of estrone and estradiol. However, all ER negative tumors contained measurable amounts of both estradiol and estrone. Higher levels of estrone were observed in ER negative tumors which correlates well with high 17 beta-hydroxysteroid dehydrogenase activity. These results suggest that false negative receptor assays in the premenopausal women is not likely to be due to occupancy of receptors by endogenous estrogens. Furthermore, the higher estrone content in the ER negative group is probably due to high 17 beta-hydroxysteroid dehydrogenase activity inherent to these tumor cells.     

Breast infiltrating ductal carcinoma: analysis of hormone, HER-2 receptors and Ki-67 proliferation marker

The aim of this study was to analyse breast carcinomas with discordant receptor status, probably hormonal dependent (estrogen receptor (ER) positive, progesterone receptor (PR) negative or ER-PR + subgroup profile) infiltrating ductal breast carcinomas not otherwise specified (IDC NOS). Specimens from 90 IDC NOS were grouped into three categories according to hormonal status: dependent (D) (ER +PR +), probably dependent (PD) (ER +PR- or ER-PR +) and non-dependent (ND) (ER-PR-); they were evaluated considering some established prognostic parameters in breast carcinomas. Statistically significant difference was found between tumor receptor status distribution and menopausal status (p = 0.0235), age of the patients (p = 0.000467), histological grade (p = 0.000003), vascular invasion (p = 0.006), HER-2 status (p = 0.0039) and Ki-67 proliferation rate (p = 0.000311). D tumors were found exclusively in post-menopausal patients (average age 68.9 years), most of which had intermediate (II) grade, without vascular invasion, with HER-2 status score predominantly 0 or 1 + and lower Ki-67 proliferation rate. PD tumors were found predominantly in younger post-menopausal patients (average age 57.5 years), with vascular invasion found in 23% of the cases. ND tumors mostly had higher histological grade, showed the highest percentage of the Ki-67 positive tumor cells and vascular invasion in 30% of the cases. We conclude that the patients with PD breast carcinomas were younger post-menopausal women with the tumors moderately differentiated, HER-2 score 0 or 1+ and with lower Ki-67 proliferation rate.     

Image cytometry of estrogen receptors in breast carcinomas

A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran-coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER-positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ER-ICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy.     

Computer-assisted immunocytochemical determination of breast cancer steroid receptors on cytological smears of excised surgical specimens compared with frozen sections

BACKGROUND: Due to the widespread use of fine needle aspirate biopsy the practice of determining estrogen (ER) and progesterone (PR) receptors in breast carcinoma from cytological smears (CS) is becoming very common. The aim of this study was to determine concordance between ER and PR assessed by immunocytochemical assay (ICA) on CS and FS both evaluated by image analysis since we have found no data in literature on this. METHODS: 104 breast carcinoma cases were selected. For all cases ER and PR determination was performed on CS, obtained by light scraping of the freshly cut surface of the excised surgical tumors at the time of frozen section diagnosis, and FS using the same monoclonal antibodies. Computer-assisted image analysis was performed in all cases using CAS 200. Results were expressed as percent positive area of neoplastic nuclei compared with total nuclear area of the examined neoplastic cells. RESULTS: Good correlation was demonstrated between percent positive nuclear neoplastic area by ER-ICA on CS and FS (r = 0.759; P < 0.0001). Concordance of results was 90.19% (P < 0.001). Good correlation was also demonstrated between percent positive nuclear neoplastic area by PR-ICA in CS and FS (r = 0.889; P < 0. 0001). Concordance of results was 97.02% (P < 0.0001). CONCLUSIONS: Our data suggest that ICA on CS with automated image analysis is efficient in evaluating ER and PR content in human breast cancer, especially when CS is the only method pathologists have to evaluate receptor status e.g. in advanced breast cancer cases when neoadjuvant therapy is necessary before surgery or when surgery is impossible.     

Claudin-16 reduces the aggressive behavior of human breast cancer cells

Claudin-16 (Paracellin-1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re-absorption of magnesium and calcium in the kidney. This study examined expression of Claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin-16 levels and patient prognosis. Insertion of the Claudin-16 gene into MDA-MB-231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and "tighter" colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno-stained. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, normalized with cytokeratin-19 levels). Levels of expression of Claudin-16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin-16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type.     

ER/PR阳性和阴性乳腺癌的定量蛋白质组学和生物信息学比较研究

目的:建立雌/孕激素受体(ER/PR)阴性和阳性乳腺癌的蛋白质表达谱,寻找ER/PR阴性和阳性乳腺癌中差异表达蛋白,为乳腺癌患者提供新的预后预测指标和治疗新靶点。方法:应用蛋白质组学i TRAQ技术建立ER/PR阳性和阴性乳腺癌的蛋白质差异表达谱,鉴定两组乳腺癌的差异表达蛋白,对部分差异表达蛋白进行生物信息学分析,包括蛋白功能注释和分类GO分析和KEGG通路分析。结果:应用i TRAQ蛋白质组学技术对乳腺癌组织进行了蛋白组学分析,鉴定出ER/PR阳性和阴性组间有差异表达的蛋白4999种,以ER/PR阳性:ER/PR阴性≥3为上调标准,确定ER/PR阳性组上调蛋白101种。以ER/PR阳性:ER/PR阴性≤0.5为下调标准,ER/PR阳性组下调蛋白122种。GO分析结果显示ER/PR受体阴性和阳性乳腺癌的差异表达蛋白的分子功能、生物过程、细胞定位较为复杂,并且在上调蛋白和下调蛋白上存在分布差异。KEGG通路分析发现部分差异表达蛋白涉及201条信号通路。结论:ER/PR阳性和阴性乳腺癌间存在差异表达蛋白,这些蛋白涉及复杂的分子功能、生物过程和信号通路。     

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.     

Comparison of visual and CAS-200 quantitation of immunocytochemical staining in breast carcinoma samples.

Using the CAS-200 image analysis system, we compared the relationship between semiautomated computer measurements (QIC score) and visual scoring (H score) on 30 breast cancer fine needle aspirates (FNAs) and corresponding tissue specimens stained by the estrogen receptor immunocytochemical assay. These results were compared to the corresponding biochemistry assays for ER. We also investigated whether the computerized system could decrease false-negative staining for ER in 32 cryostat sections. QIC scores were generated using fixed nuclear and antibody thresholds after standardizing the illumination. Computer quantitation was essentially as precise as visual semiquantitation for FNAs: a small but significant correlation was found between tumor ER content and QIC score (r = .504, P less than .02) as compared with H score and ER content (rs = .55, P less than .005). The computerized system did not decrease the false-negative rate in cryostat sections. In all, computerized quantitation was no better than visual analysis of ER staining in these breast carcinomas.     

A combined histological, immunocytochemical and biochemical approach in the evaluation of estrogen receptors in breast carcinomas

Correlation of structural and functional data might lead to better identification of hormone-dependent tumors. Sixty breast cancer specimens, sent to the biochemistry laboratory for estrogen receptor (ER) analysis, were studied here by a combined morpho-functional approach. Histological examination of needle biopsies on frozen tissue blocks showed that 12 cases (10%) were free of tumor cells; these cases mostly proved ER negative. On the other 48 cases, an immunocytochemical reaction was performed on the biopsy sections with a monoclonal antibody directed against p 29, an estrogen receptor related antigen. The staining values for p 29 and the biochemical ER findings were significantly correlated. A combined histological, immunocytochemical study seems to offer advantages in the selection of patients for hormonal therapy.     

Epidermal growth factor receptors in human breast and endometrial carcinomas

The incidence and levels of epidermal growth factor receptor (EGFR) were studied in 67 breast tumors and 22 endometrial carcinomas. Estrogen receptors (ER) were also measured in all samples and progesterone receptors (PR) were analyzed in 57 breast samples and 21 endometrial tumors. A high level of EGFR expression is found in both breast and endometrial carcinomas, although the incidence of EGFR content is greater in breast carcinomas. 36% of breast tumors had EGFR at levels 3-49.5 fmol/mg membrane protein, whereas this percentage of positivity was 27% for endometrial tumors. In 51% of breast carcinoma and 73% of endometrial tumors, there was a positive ER content, whereas 53% of breast tumors and 62% of endometrial carcinomas were positive. A clear inverse relationship between EGFR content and ER and PR status has been observed in breast tumors. Our data confirm the previously described inverse correlation between expression of EGFR and estrogen receptors in human breast cancer. We also show here that there is a similar inverse relationship between EGFR content and ER levels in endometrial tumors.     

Steroids and human breast cancer.

Normal mammary gland cells are sensitive to a number of hormones, of which estrogen and prolactin exert the most obvious effects. Some breast cancer cells are also sensitive. Cytoplasmic receptor sites for each hormone are responsible for the interaction between the hormone and the cell. The presence of estrogen receptor has been especially studied in humans. Data collected from several sources are reviewed. The prese nce of estrogen receptors has been assayed in 154 primary breast tumors and 72 metastatic breast tumors for correlation with response to endocri ne therapy. Positive values were found in 70% of primary and 58% of metastatic specimens. Of 211 treatment trials, ablative therapy produced objective tumor regressions in 33%. Of the 94 trials with negative receptor values, only 8 were successful while 59 of the 107 trials in patients with positive receptor values succeeded. In those with borderline tumor receptor, values had a 30% response. With additive therapy, 34% of 170 trials showed tumor regression. Of these, 82 had negat ive receptor values but 8% were successful, whereas of 85 with positive receptor values, 60% were favorable. With miscellaneous therapy, 27% of 55 trials gave responses to a variety of endocrine therapies, including antiestrogens. The 32 with negative receptor values gave 16% of favorable responses whereas 43% of 23 trials in those with positive receptor values succeeded. Estrogen receptor assays performed routinely would spare patients with negative results from unnecessary major ablative therapy. Of those with positive findings, 55-60% might be benefited. The fact that all with positive receptor values do not respond is attributed to the fact that this is only part of the hormonal control system. Other biochemical lesions are assumed to have occurred in patients when endocrine therapy fails despite positive estrogen receptor levels as measured.     

Image cytometry of aneuploidy, growth fraction (MoAb Ki-67) and hormone receptors (ER, PR) immunocytochemical assays in breast carcinomas

DNA nuclear content was assessed in human breast carcinomas (n = 132) using image cytometry. Optical density histograms of Feulgen stained cell imprints from fresh tissue samples, subsequently frozen for immunocytochemical assays, were determined by the SAMBA system and used for the DNA index, the ploidy balance (PB) and the proliferation index (PI) computation. The three parameters were correlated to (i) histological data (tumour grade, vascular and/or lymph node invasion) and to (ii) growth fraction (Ki67), hormone receptor antigenic sites (ER, PR) and intramedullar (bone marrow) biopsies and anti-KL1-positive epithelial cells. It was shown that 57% of breast carcinomas were aneuploid. Aneuploidy PI significantly correlated to the criteria of poor prognosis such as high tumour grade, vascular and lymphatic invasion and to increased Ki67-positive cells, and the absence of or low ER and PR. Since image cytometry is easy to handle and perfectly suitable for current diagnostic practice in pathology departments, particularly for tumour cell ploidy assessment and standardized analysis of immunostaining procedures with morphological control of the preparation, we conclude that image cytometry, as performed with the SAMBA, must be regarded as a relevant tool for prognosis evaluation and therapy guidance in individual patients.     

Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma.

The assessment of apoptosis in solid tumors is of interest because of its biological role in tumor evolution and response to therapy. A commonly used method for apoptosis measurement is the TUNEL 3' end-labeling technique, which has shown wide variations in results when applied to solid tumors. Thirty-one fine needle breast carcinoma samples were analyzed by fluorescent TUNEL assay and DNA content using image analysis and flow cytometry. TUNEL positivity, seen both in cells with apoptotic morphology and in a subset of morphologically normal cells, was categorized into five staining patterns and quantitated. Values for patterns of TUNEL-positive cells were compared with TUNEL positivity measured by flow cytometry. Flow cytometric quantitation showed a mean of 24.3% positive cells, which correlated (P < 0.02) with total positive cells (all patterns) measured by image (22.4%). Image analysis quantitation of morphologically apoptotic cells (4.2%) did not correlate with flow cytometric TUNEL positivity and the majority of TUNEL-stained cells were morphologically normal (17%). Image analysis allows discrimination of TUNEL-positive morphologically apoptotic and nonapoptotic cells, which are included in the total number of TUNEL-positive events measured by flow cytometry.