Tumor Estrogen Receptor Content Research Articles

Abstract 673: Exploration of tissue morphologies in breast cancer samples using unsupervised machine learning

Abstract We applied a machine learning approach for exploration of tissue morphology in hematoxylin and eosin (H&E) stained breast cancer tissue microarray (TMA) samples. We then investigated whether the morphological categories produced were associated with clinically relevant molecular biomarkers and 10-year overall survival. The data set comprises digitized (0.22 µm/pixel) and H&E stained TMA spots from tumor samples of 490 women who were diagnosed with primary breast cancer within a Finnish breast cancer database (FinProg) collected in 1991 and 1992. In order to quantitatively describe the tissue morphologies of the TMA spots, we divided the tissue images into rectangular sub-images (224x224 pixels), and extracted features with a pre-trained convolutional neural network. We then clustered the sub-images (n=147,266) with a non-linear data embedding algorithm that creates a two-dimensional mapping of the tissue morphologies. Lastly, we defined a quantitative profile for each tumor, describing the morphologies within the tissue spot image by dividing the two-dimensional map of morphologies into 128 separate clusters with k-nearest neighbor clustering. Visual inspection of the two-dimensional embedding of tissue spot images verified that the morphologies clustered coherently, i.e. similar looking sub-images formed distinct clusters in the map. Interestingly, some morphological patterns were strongly associated with tumor estrogen receptor content, progesterone receptor content, human epidermal growth factor receptor 2 status, and the proliferation marker Ki-67 status (p<0.0001 for each comparison). In exploratory analyses we identified one morphological category that was associated with a favorable 10-year overall survival with a risk ratio of 0.68 (CI95% 0.53-0.89, p=0.002, power = 0.87). Our work demonstrates that unsupervised machine learning can be applied to explore and better understand the role of morphological patterns in breast cancer. Methods that quantitatively assess the morphology of cancer tissue may complement molecular biomarkers and potentially reveal novel prognostic and predictive factors. Citation Format: Riku Turkki, Dmitrii Bychkov, Nina Linder, Jorma Isola, Heikki Joensuu, Johan Lundin. Exploration of tissue morphologies in breast cancer samples using unsupervised machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2017-673

Localization of metallothionein in breast carcinomas. An immunohistochemical study.

Metallothionein (MT) is a cysteine-rich, low molecular weight protein that binds zinc, copper, and cadmium. It is present in a number of normal cells including hepatocytes particularly during fetal and early postnatal life. It has been suggested that developmental profile of MT is similar to other oncofetal gene products and hence, it could be used as a marker for aggressive tumour behaviour. In order to test that hypothesis, we used a monoclonal antibody to MT and immunohistochemically evaluated formalin-fixed, paraffin-embedded tissues from 79 breast carcinomas. In non-neoplastic breast tissue, a strong nuclear and cytoplasmic staining was observed in myoepithelial cells. Positive staining for MT was present in 35 (44%) of breast carcinomas. In most positive cases, nuclear, or both nuclear and cytoplasmic staining was seen. All positive tumours were invasive ductal carcinomas, including a medullary and a metaplastic carcinoma. None of the mucinous, lobular, or intraductal papillary carcinomas reacted for MT. A statistically significant association was found between MT immunostaining and histological grade (P < 0.01) as well as with nuclear grade (P < 0.01). We also observed an inverse relationship between MT staining and oestrogen receptor content of tumours (P < 0.01). Similarly, a statistically significant association was found between moderate and strong MT immunostaining and decreased overall survival and shorter disease-free survival (P < 0.01). MT immunostaining was also predictive of a worse prognosis in the subgroup of lymph node negative (P < 0.001) and oestrogen receptor negative patients (P < 0.01). No statistically significant association was found between MT staining and size of tumour or the presence of lymph node metastasis. We conclude that MT staining may be a useful marker of less differentiated and more aggressive carcinomas of the breast.

Comparison of Tritiated Estradiol and Tamoxifen Aziridine for Measurement of Estrogen Receptors in Human Breast Cancer Cytosolss

We examined the estrogen receptor measurement in 265 human breast cancer cytosols by using a specific method based on [3H]tamoxifen aziridine labeling, sequential immunoadsorption with an antiestrogen receptor monoclonal antibody (H-222), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These new tools of molecular endocrinology revealed an impressive estrogen receptor molecular polymorphism. Given the recent finding of a similar estrogen receptor polymorphism at the messenger RNA level by several laboratories, it is tempting to speculate about its possible biological significance. To gain insight into the potential clinical relevance of this polymorphism in terms of breast cancer hormone dependence, we compared the 265 cytosols for their [3H]tamoxifen aziridine- and [3H]estradiol-binding capacities using the above-mentioned method and the conventional dextran-coated charcoal assay. We failed to identify a specific [3H]tamoxifen aziridine electrophoretic pattern with respect to the tumor estrogen receptor content as measured by the dextran-coated charcoal assay. However, an excellent correlation overall was found between the intensities of both labeling methods. Some tumors were positive for only one of these two ligands. It will be clinically important to see whether the tumors positive for [3H]tamoxifen aziridine only correspond to the small subset of tumors (10%) which respond to tamoxifen treatment despite very low estrogen receptor levels, as measured by the dextran-coated charcoal technique.

Pineal melatonin inhibition of tumor promotion in theN-nitroso-N-methylurea model of mammary carcinogenesis: potential involvement of antiestrogenic mechanisms in vivo

The N-methyl-N-nitrosourea (NMU) model of hormone-responsive rat mammary carcinogenesis was used to address the hypothesis that melatonin (Mel), the principle hormone of the pineal gland, inhibits tumorigenesis by acting as an anti-promoting rather than an anti-initiating agent. Daily late-afternoon injections of Mel (500 micrograms/day), restricted to the initiation phase of NMU mammary tumorigenesis, were ineffective in altering tumor growth over a 20-week period. When Mel treatment was delayed for 4 weeks after NMU and then continued through the remainder of the promotion phase, only tumor number was significantly lower than in controls. However, when Mel injections encompassed the entire promotion phase, both tumor incidence and number were significantly lower than in the controls. Although elimination of the endogenous Mel signal via pinealectomy promoted tumor growth, the effect was not statistically significant. Serum levels of estradiol and tumor estrogen receptor content were unaltered by either Mel or pinealectomy. While Mel treatment failed to affect circulating prolactin levels, pinealectomy caused a two-fold increase in serum prolactin. The estradiol-stimulated recrudescence of tumors following ovariectomy was completely blocked by either 20, 100 or 500 micrograms Mel/day or tamoxifen (20 micrograms/day). Thus, Mel appears to be an anti-promoting hormone that may antagonize the tumor-promoting actions of estradiol in this model of mammary tumorigenesis.

Immunohistochemical Expression of c-erbB-2 in Human Breast Cancer by Monoclonal Antibody: Correlation with Lymph Node and Er Status

c-erbB-2 Protein expression was investigated in a series of fifty primary breast cancers by means of a specific monoclonal antibody and immunocytochemistry. Specific staining was observed at the plasma membrane level of neoplastic cells, according to the reported localization of c-erbB-2 protein. Sixty-four percent of tumors scored positive, with a variable amount of stained cells. The rate of protein expression was found to exceed the reported gene amplification. No relationship was observed between c-erbB-2 protein staining and age, menopausal status or histologic subtypes. An inverse association was found between c'erbB-2 protein staining and estrogen receptor content of tumors, assayed by immunocytochemistry. A positive relationship was observed between c-erbB-2 protein expression and presence of axillary node metastasis. These findings suggest that c-erbB-2 protein expression is a marker of tumor aggressiveness and that its prognostic power deserves further investigation both in node-positive and node-negative patients.

Breast carcinoma cellularity and its relation to oestrogen receptor content.

The cellularity of 104 primary breast carcinomata was determined by semiautomated image analysis to allow the relation between cellularity and oestrogen receptor content values to be assessed. The oestrogen receptor content of tumours detected by a steroid binding assay showed no correlation with cellularity, although a possible weak negative correlation was observed between tumour cellularity and oestrogen receptor content detected by enzyme immunoassay. It is concluded that there is no single direct correlation between tumour cellularity and oestrogen receptor content.

Characterization of the uptake of 16α-([ 18F]fluoro)-17β-estradiol in DMBA-induced mammary tumors

In order to investigate possible correlations between the uptake of 16α-([ 18F]fluoro)-17β-estradiol ( 18F-ES) by 7,12-dimethylbenz(a) anthracene (DMBA)-induced tumors in rats and the estrogen receptor (ER) content of these tumors, a comprehensive study was performed in which the tissue distribution of 18F-ES was measured in tumor-bearing rats, together with simultaneous measurements of blood volume (by technetium-labeled red blood cells) and blood flow (by iodoantipyrine infusion). In addition, the time course of 18F-ES metabolism and the tissue distribution of the metabolites was studied. Metabolism of 18F-ES is very rapid, and after 2 h, most of the activity in blood and nontarget tissues is due to metabolites; target tissue activity, however, is due mainly to unmetabolized compound. Most of the circulating activity, both 18F-ES and its metabolites, is strongly associated with macromolecules or cells, and while the metabolites are not taken up selectively by target tissues, they do enter nontarget tissues. Tumor blood volume and blood flow vary widely, but not in a way that appears related to tumor necrosis. The uptake of 18F-ES by the uterus and DMBA-induced mammary tumors of adult rats reaches maximum levels ( ca 0.35 and 0.10% I.D./g·kg, respectively) at early times (0–1 h), and drops slowly thereafter. The uterus to nontarget or tumour to nontarget tissue ratios, however, start low and continue to increase, reaching maximum levels ( ca 20 and 15, respectively) at 2–3 h. There does not, however, appear to be a simple relationship between tumor uptake (either as % I.D./g·kg or tumor to nontarget ratio) measured at a single 3 h time point and tumor ER content, even considering differences in tumor blood flow. This suggests that an estimation of tumor ER content will require the application of more complex pharmacodynamic models that involve the measurement of the complete profile of receptor lignad uptake, retention, and washout from target to nontarget areas. The application of such models will be assisted by the development of estrogen receptor binding ligands that are not converted to circulating metabolites.

Relationship between mammographic pattern and estrogen receptor content in breast cancer.

The mammographic pattern and its relationship to the estrogen receptor (ER) content was studied in 184 breast cancers. The tumors were divided into five radiographic subgroups: mass with spicules (A), diffuse (B), clusters of calcifications without a mass (C), circumscribed (D), and not visible at mammography (E). The ER content of tumors belonging to group A was higher than that of the other subgroups in both pre- and postmenopausal women. 121 tumors belonged to group A, of which 80% were ductal cancers. The tumors in groups B and C had very low ER values and those of groups D and E had intermediate values. The likelihood of finding a high estrogen receptor content of a tumor is thus greater when the tumor radiographically is seen as a mass with spicules than when seen as an increased attenuation or as only clusters of calcifications. It is suggested that patients with tumors belonging to group A should have a more favorable prognosis than patients with tumors belonging to groups B and C.

Effects of reserpine administration on rat mammary tumors and uterine disease induced by N-Nitrosomethylurea

Reserpine 50 μg 100 g body weight administered s.c. to female rats on the day before, of and after each of three N-nitrosomethylurea (NMU) i.v. injections, influenced a number of experimental observations. The total doses of NMU given to the various treatment groups were 15, 12, 9, 6, 3 and 1.5 mg 100 g body weight. Tumor incidence ranged from approximately 100 to 0% in the control and reserpine-treated groups, depending on the dose of carcinogen given. Concurrent reserpine administration with the NMU decreased the number of tumors per rat; at 15 and 12 mg 100 g body weight NMU doses the values were only 58 and 55% of the respective controls. Reserpine treatment at these 2 NMU doses also increased the percentage of well-differentiated mammary tumors compared with corresponding controls. Tumor estrogen receptor levels in controls ranged from 71 to 78 fmol/mg protein and in the reserpine-treated groups from 96 to 105 fmol/mg protein; significant elevations were present in the rats given reserpine and the 2 highest doses of NMU. Tumor progesterone and prolactin receptor levels were similar in control and reserpine-treated groups. The estrous cycle of the controls was frequently arrested at estrus and 21 57 (37%) had a fluid-distended uterus and thickened uterine walls; only 2 52 (4%) of reserpine-treated rats had hydrometria and none exhibited cycle arrest at estrus. The effect of reserpine on mammary tumors was also studied when given after initial exposure to the carcinogen. NMU was administered to 65 rats in 3 doses of 5 mg 100 g body weight by i.v. injections 4 weeks apart. Equal numbers of animals comprised 1 control and 2 reserpine treatment groups. Reserpine was given at a daily dose of 20 μg 100 g body weight commencing 67 days after the first NMU dose (chronic regimen) or by the same schedule preceded by 50 μg 100 g doses given on the day before, with and on the day after the second and third NMU injections (acute/chronic regimen). A fourth group of rats received chronic reserpine treatment but no NMU. All 3 NMU-exposed groups had a 100% tumor incidence; none developed in the reserpine-treated controls. The acute/chronic reserpine treatment increased the rate of tumor development but neither drug schedule altered the number of tumors per rat or the mean final total tumor areas. Reserpine administration was associated with greater degrees of anaplasia in mammary tumors induced by NMU ( P < 0.01). The mean tumor estrogen receptor content was significantly higher in the acute/chronic reserpine-treated group ( P < 0.0002) and the chronic reserpine-treated animals ( P < 0.008) compared with controls. Tumor prolactin receptor content was unaffected by the drug. Peroxidase activity was higher in mammary tumors and lower in uteri from reserpine-treated rats compared with tumor-bearing controls. Also, there was a strong correlation ( r = 0.82) between the mammary tumor estrogen receptor and tumor peroxidase activity for rats treated with the acute/chronic reserpine regimen.