Invasive Mammary Cancer Research Articles

Assessment of Tissue Eosinophilic Infiltration in Invasive Mammary Carcinoma.

Background: Stromal inflammatory cells in malignant tissue have recently gained increasing interest. Unlike the extensive research on tumor-infiltrating lymphocytes, published data about tumor-infiltrating eosinophils in breast cancer are scarce. Furthermore, similar studies have yet to be conducted in Iraq. Aims: The objective of this study is to examine the presence of eosinophilic infiltration by direct visualization using light microscopy and to analyze its relationship with other histological parameters in a group of Iraqi women diagnosed with invasive mammary cancer. Methods and material: A retrospective study enrolled 90 histological samples of invasive mammary carcinoma provided by core needle biopsy from a single center, together with their immunohistochemical results for ER and HER2-NEU. Data reviewing, direct morphological visualizations, and counting eosinophilic infiltration in tissue sections were done by two independent pathologists using light microscopy. The results were statistically correlated with the grade, ER, HER2-NEU, calcification, and axillary lymph node status at presentation. Results: Out of the entire sample size (90), 40 (44%) showed the presence of eosinophilic infiltration in the tissue, both intratumoral and stromal. Further analysis revealed that most eosinophilic infiltrates had an intermediate score (4-19) per 10 consecutive high-power fields. A strong and meaningful statistical relationship was seen between tissue eosinophilic infiltration and HER2/NEU status. A statistically insignificant correlation was seen between tissue eosinophilic infiltration and histological grade, ER receptor status, calcification, and axillary lymph node status at presentation. Conclusions: Eosinophils are tumor-infiltrating cells in breast cancer, both intratumoral and stromal. The presence of tissue eosinophilic infiltration can predict HER2/NEU negativity in breast cancer.

Suppression of mTOR Expression by siRNA Leads to Cell Cycle Arrest and Apoptosis Induction in MDA-MB-231 Breast Cancer Cells.

Mammary carcinogenesis, being ranked second in cancer-related mortality and the inadequacy of existing chemotherapy advocates the development of a novel treatment approach targeting its molecular signalling. Hyperactivation of mammalian target of rapamycin (mTOR) has a critical role in developing invasive mammary cancer and it can be a potential target. This experiment was to explore the efficacy of mTOR-specific siRNA on therapeutic targeting of the mTOR gene, assess its proficiency in suppressing in vitro breast cancer and determine underlying molecular mechanisms. Specific siRNA targeting mTOR was transfected into MDA-MB-231 cells and mTOR downregulation was validated through qRT-PCR and western blot analysis. Cell proliferation was analysed by MTT assay and confocal microscopy. Apoptosis was studied through flow cytometry and S6K, GSK-3β and caspase 3 expression were estimated. Further, the effect of mTOR blockade on cell cycle progression was determined. Following transfection of mTOR-siRNA into the MDA-MB-231 cells, cell viability and apoptosis were examined which indicates that clinically relevant concentration of mTOR-siRNA inhibited cell growth and proliferation and promote apoptosis, resulting from the suppression of mTOR. This leads to the downregulation of mTOR downstream S6K and upregulation of GSK-3β. An increased level of caspase 3 symbolises that the apoptotic activity is mediated through caspasedependent pathway. Further, mTOR downregulation causes cell cycle arrest in G0/G1 phase as observed in the flow cytometry study. With these results, we can conclude that mTOR-siRNA exerts direct 'anti-breast cancer' activity propagated by the S6K-GSK-3β- caspase 3 mediated apoptosis and by inducing cell cycle arrest.

Expression of fascin in Invasive mammary cancer - A study at a Tertiary care centre in Southern India

Introduction and Aim: Breast cancer is the commonest malignancy in women. Most women do not die of the primary tumor but from metastasis and local invasion. Augmented motility of cancer cells corroborates with greater metastatic potential, that is brought about by actin cytoskeleton. Fascin, a cytoplasmic F-actin-bundling protein that cross-links actin filaments and its overexpression is strongly associated with metastatic progression and poor prognosis. The present study aims to determine the fascin protein expression by immunohistochemistry in invasive breast carcinoma and to correlate the same with the existing prognostic factors. Methods: Immunohistochemical expression of fascin in 100 cases of invasive mammary carcinoma was studied. Data collected was scored based on intensity and statistically analyzed using GNU-PSPP version 0.10 software. To determine significant clinico-pathological differences between fascin expression in positive and negative tumors, Pearson Chi-square test was used. Results: It was noted that 22 of the 100 cases were positive with a score of 3 or more. A significant association of nodal status with fascin (p value <0.05) was noted. Higher proportion of fascin positive tumors were node negative. There was an inverse correlation between ER/PR/HER2 status and fascin positivity. The corresponding p values for ER, PR and HER2 with fascin were <0.05. On the contrary 12(57.15%) of the 21 triple negative cases were fascin positive. The p value was <0.001 indicating a significant correlation between molecular subtypes and fascin expression. A more of the fascin positive tumors fell in the triple negative category. Conclusion: Higher proportion of fascin positive cases in the node negative category, an inverse correlation between fascin expression and individual ER/PR, HER2 expression and larger number of fascin positive cases in the triple negative category. These findings point an association of fascin with aggressive breast carcinomas.

S1746 An Unusual Case of Metastasis to Colon From Primary Breast Cancer With Subtle Colonoscopy Findings

INTRODUCTION: Breast cancer is the most common malignancy in women all over the world. Distant metastasis is by far the most common cause of death. It is common for breast cancers to metastasize to bone, liver and lung. Metastasis to the GI tract especially colon is very rare and can mimick primary colon cancer vs colitis with variable colonoscopy findings. CASE DESCRIPTION/METHODS: A 72 years old female was diagnosed with estrogen receptor-positive, well-differentiated, invasive mammary cancer in 2013. She underwent a localized lumpectomy with sentinel lymph node dissection. Her disease was stage 1A(pT1c pN0(i-) pMX). She received adjuvant radiation, tamoxifen and an aromatase inhibitor. Five years later, she was diagnosed with widespread metastatic disease to the axial and appendicular skeleton. She got palliative radiation and 10 cycles of Fulvestrant chemotherapy. A restaging CT scan 6 months later showed colonic wall thickening at the hepatic flexure. A colonoscopy showed normal colon with a subtle decrease in the mucosal vascular pattern at the hepatic flexure, biopsies were taken and pathology revealed expansion of the lamina propria by metastatic adenocarcinoma. The specimen was reviewed alongside the previous biopsy of the T9 vertebral lesion and showed similar histologic features, compatible with metastases from the patient's known breast primary. In addition, CK7 was positive and CK20 was negative. In the subsequent 8 months, she received 8 cycles of chemotherapy but developed worsening abdominal pain, inability to tolerate diet and an interval CT scan showed progression of colonic wall thickening in ascending colon and proximal transverse colon. Shortly after, she passed away from her widespread metastatic disease. DISCUSSION: The incidence of breast cancer metastasizing to GI tract is 3.4-4.5%, according to one study, 92% of it involves stomach. Metastasis from primary breast cancer to the colon is extremely rare. Usually liver, bone and lungs are the most common sites of metastasis. Presentation is variable and symptoms include abdominal pain, weight loss, bowel obstruction, anemia and GI bleed. Certain markers such as CK7 can be used to differentiate between primary GI cancers and metastasis from breast cancer. Diagnosis can be challenging as these colonic lesions can mimic other diseases such as IBD, radiation colitis and primary GI cancers. Colonoscopy findings can be variable and as in this case, even minimal changes in background of known malignancy warrants a biopsy for early diagnosis.Figure 1.: Decreased mucosal vascular pattern at the hepatic flexure.

Responsiveness of Brca1 and Trp53 Deficiency-Induced Mammary Preneoplasia to Selective Estrogen Modulators versus an Aromatase Inhibitor in Mus musculus.

An intervention study initiated at age 4 months compared the impact of tamoxifen (25 mg), raloxifene (22.5 mg), and letrozole (2.5 mg) administered by 60-day release subcutaneous pellet on mammary preneoplasia prevalence at age 6 months in conditional genetically engineered mouse models with different Breast cancer 1 (Brca1) gene dosages targeted to mammary epithelial cells and germline Tumor protein P53 (Trp53) haploinsufficiency (10-16/cohort). The proportion of unexposed control mice demonstrating mammary preneoplasia at age 6 months was highest in Brca1fl11/fl11/Cre/p53-/+ (54%) mice followed by Brca1WT/fl11/Cre/p53-/+ mice (30%). By age 12 months, invasive mammary cancers appeared in 80% of Brca1fl11/fl11/Cre/p53-/+ and 42% of Brca1WT/fl11/Cre/p53-/+ control unexposed mice. The spectrum of cancer histology was similar in both models without somatic mutation of the nongenetically engineered Brca1, Trp53, Brca2, or Death-associated protein kinase 3 (Dapk3) alleles. Two-month exposure to tamoxifen, raloxifene, and letrozole significantly reduced estrogen-mediated tertiary branching by 65%, 71%, and 78%, respectively, in Brca1fl11/fl11/Cre/p53-/+ mice at age 6 months. However, only letrozole significantly reduced hyperplastic alveolar nodules (HAN) prevalence (by 52%) and number (by 30%) and invasive cancer appeared despite tamoxifen exposure. In contrast, tamoxifen significantly reduced HAN number by 95% in Brca1WT/fl11/Cre/p53-/+ mice. Control mice with varying combinations of the different genetically modified alleles and MMTV-Cre transgene demonstrated that the combination of Brca1 insufficiency and Trp53 haploinsufficiency was required for appearance of preneoplasia and no individual genetic alteration confounded the response to tamoxifen. In summary, although specific antihormonal approaches showed effectiveness, with Brca1 gene dosage implicated as a possible modifying variable, more effective chemopreventive approaches for Brca1 mutation-induced cancer may require alternative and/or additional agents. Cancer Prev Res; 10(4); 244-54. ©2017 AACR.

Correlation of In Vivo and Ex Vivo ADC and T2 of In Situ and Invasive Murine Mammary Cancers.

Ex vivo MRI may aid in the evaluation of surgical specimens, and provide valuable information regarding the micro-anatomy of mammary/breast cancer. The use of ex vivo MRI to study mouse mammary cancer would be enhanced if there is a strong correlation between parameters derived from in vivo and ex vivo scans. Here, we report the correlation between apparent diffusion coefficient (ADC) and T2 values measured in vivo and ex vivo in mouse mammary glands with in situ cancers (mammary intraepithelial neoplasia (MIN)) and invasive cancers (those which spread outside the ducts into surrounding tissue). MRI experiments were performed on the Polyoma middle T oncoprotein breast cancer mouse model (n = 15) in a 9.4T scanner. For in vivo experiments, T2-weighted (T2W) images were acquired to identify abnormal regions, then ADC and T2 values were measured for nine selected slices. For ex vivo experiments, a midline incision was made along the spine, and then skin, glands, and tumors were gently peeled from the body. Tissue was fixed in formalin, placed around a mouse-sized sponge, and sutured together mimicking the geometry of the gland when attached to the mouse. The same pulse sequences used for in vivo experiments were repeated for ex vivo scans at room temperature. Regions of interest were manually traced on T2W images defining features that could be identified on in vivo and ex vivo images. The results demonstrate a strong positive correlations between in vivo and ex vivo invasive cancers for ADC (r = 0.89, p <0.0001) and T2 (r = 0.89, p <0.0001) values; and weak to moderate positive correlations between in vivo and ex vivo in situ cancers for ADC (r = 0.61, p <0.0001) and T2 (r = 0.79, p <0.0001) values. The average ex vivo ADC value was about 54% of the in vivo value; and the average ex vivo T2 was similar to the in vivo value for cancers. Although motion, fixation, and temperature differences affect ADC and T2, these results show a reliable relationship between ADC and T2 in vivo and ex vivo. As a result ex vivo images can provide valuable information with clinical and research applications.

Abstract 2266: Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair

Abstract According to the American Cancer Society, more than 90% of breast cancer patients who succumb to the condition die as a result of the cancer metastasizing to other areas of the body. Our lab has previously shown that the presence of a chemoresistant invasive subpopulation of breast carcinoma cells is correlated with the prevalence of metastasis. Additionally, we have hypothesized that chemoresistance and survival of these cancer cells which eventually leads to relapse and metastasis following initial treatment with chemotherapeutic agents is achieved by enhanced DNA repair capability in this population. We reported that an anti-apoptotic protein ARC (apoptosis repressor with caspase recruitment domain) is over expressed in the invasive mammary cancer cells that are resistant to standard chemotherapeutic agents. Further, we have shown that ARC over expressing cells are resistant to chemotherapy both in vivo and in vitro. In this study, we examined cellular DNA repair signaling pathways in order to delineate the role of ARC in the pathogenesis of breast cancer. Utilizing cell lines where ARC was either knocked down or overexpressed, we investigated potential correlations of ARC with DNA repair capability by means of the single cell gel electrophoresis assay (comet assay). The results of these experiments indicated that over expression of ARC leads to increased cellular DNA repair while knocking it down leads to decreased cellular DNA repair. In order to determine the molecular basis of the role of ARC in the DNA repair pathways, we have performed qRT-PCR based gene expression analysis on ARC modified cell lines using techniques previously established in our lab. The results of these experiments indicated that increased levels of ARC are correlated with upregulation of key genes in single stranded DNA repair pathways such as the base excision and nucleotide excision repair pathways. We are working to identify the specific domain of ARC that serves as its effector in the DNA repair pathways. In particular, we are looking at the caspase recruitment domain (CARD) of ARC which is known to be key for ARC`s function in the apoptotic pathways. Once this domain is identified, it may serve as a potential drug target, the silencing of which could result in the disruption of key pathways required for invasive breast cancer cell survival. Such therapy may facilitate the ablation of chemoresistant breast cancer cell populations, prevent metastasis and tumor relapse, and increase long term patient survival. Citation Format: Eli Grunblatt, Evanka Madan, Sweta Roy, Sumanta Goswami. Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2266. doi:10.1158/1538-7445.AM2014-2266

Abstract 175: Prevention of mammary carcinogenesis and pulmonary metastasis by Ayurvedic medicine constituent withaferin A in a clinically-relevant transgenic mouse model.

Abstract Novel strategies for prevention of breast cancer are desirable mainly because many risk factors associated with this disease are not easily modifiable. In addition, currently available clinical options for prevention of breast cancer (eg, selective estrogen receptor modulators and aromatase inhibitors) are sub-optimal. Recent literature highlights merit of plants used in Ayurvedic medicine, which has been practiced for centuries in India for treatment of different disorders, for identification of novel cancer preventive agents. Withania somnifera (also known as Ashwagandha) is one such medicinal plant that is used to alleviate a variety of symptoms. In experimental models, the known pharmacological effects of Withania somnifera include cardioprotection from ischemia reperfusion injury, inhibition of experimental Parkinsonism, suppression of hepatic lipid peroxidation, antibacterial effects, and anticancer effects. Anticarcinogenic effect of Withania somnifera is ascribed to withanolides such as withaferin A (WA). We have shown previously that WA treatment inhibits growth of cultured and xenografted human breast cancer cells by causing apoptotic cell death. The present study extends these observations and determines efficacy of WA for prevention of breast cancer in a clinically-relevant transgenic mouse model (MMTV-neu). Incidence and burden of mammary cancer and pulmonary metastasis were scored in female MMTV-neu mice after 28 weeks of intraperitoneal administration (three times/week) with 0.1 mg WA (n = 32) or vehicle (n = 29). This mild-regimen of WA administration resulted in a statistically significant decrease in macroscopic (palpable) tumor size, microscopic tumor area, and incidence of pulmonary metastasis. For example, the mean wet weight of the palpable tumor was lower by 50% in WA-treated mice compared with control mice (P = 0.03). Likewise, the area of invasive mammary cancer was lower by 95.14% in WA-treated mice compared with control mice (P = 0.05). The WA-treated mice exhibited a 72.8% decrease in incidence of pulmonary metastasis compared with the control mice (P = 0.02). Immunohistochemistry revealed reduced expression of HER-2 in tumors from WA-treated mice compared with control. Furthermore, consistent with cellular observations, mammary cancer prevention by WA treatment was associated with increased apoptosis index, inhibition of complex III activity, and increased staining for 8-oxo-dG (an indicator of oxidative stress). In conclusion, the results of the present study indicate that WA inhibits mammary carcinogenesis in a transgenic mouse model by causing apoptosis. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Eun-Ryeong Hahm, Joomin Lee, Su-Hyeong Kim, Anuradha Sehrawat, Julie A. Arlotti, Sruti Sajjan Shiva, Rohit Bhargava, Shivendra V. Singh. Prevention of mammary carcinogenesis and pulmonary metastasis by Ayurvedic medicine constituent withaferin A in a clinically-relevant transgenic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 175. doi:10.1158/1538-7445.AM2013-175

T2* relaxation times of intraductal murine mammary cancer, invasive mammary cancer, and normal mammary gland

This study investigates the feasibility of T(2)∗ to be a diagnostic indicator of early breast cancer in a mouse model. T(2)∗ is sensitive to susceptibility effects due to local inhomogeneity of the magnetic field, e.g., caused by hemosiderin or deoxyhemoglobin. In these mouse models, unlike in patients, the characteristics of single mammary ducts containing pure intraductal cancer can be evaluated. The C3(1)SV40Tag mouse model of breast cancer (n = 11) and normal FVB∕N mice (n = 6) were used to measure T(2)∗ of normal mammary gland tissue, intraepithelial neoplasia, invasive cancers, mammary lymph nodes, and muscle. MRI experiments were performed on a 9.4T animal scanner. High resolution (117 microns) axial 2D multislice gradient echo images with fat suppression were acquired first to identify inguinal mammary gland. Then a multislice multigradient echo pulse sequence with and without fat suppression were performed over the inguinal mammary gland. The modulus of a complex double exponential decay detected by the multigradient echo sequence was used to fit the absolute proton free induction decay averaged over a region of interest to determine the T(2)∗ of water and fat signals. The measured T(2)∗ values of tumor and muscle are similar (∼15 ms), and almost twice that of lymph nodes (∼8 ms). There was a statistically significant difference (p < 0.03) between T(2)∗ in normal mammary tissue (13.7 ± 2.9 ms) and intraductal cancers (11 ± 2.0 ms) when a fat suppression pulse was applied. These are the first reported T(2)∗ measurements from single mammary ducts. The results demonstrated that T(2)∗ measurements may have utility for identifying early pre-invasive cancers in mouse models. This may inspire similar research for patients using T(2)∗ for diagnostic imaging of early breast cancer.

Lysophosphatidic acid production and action: critical new players in breast cancer initiation and progression

Lysophosphatidic acid (LPA) is a potent lipid mediator that acts on a series of specific G protein-coupled receptors, leading to diverse biological actions. Lysophosphatidic acid induces cell proliferation, survival and migration, which are critically required for tumour formation and metastasis. This bioactive lipid is produced by the ectoenzyme lysophospholipase D or autotaxin (ATX), earlier known as an autocrine motility factor. The ATX–LPA signalling axis has emerged as an important player in many types of cancer. Indeed, aberrant expression of ATX and LPA receptors occurs during the development and progression of breast cancer. Importantly, expression of either ATX or LPA receptors in the mammary gland of transgenic mice is sufficient to induce the development of a high frequency of invasive and metastatic mammary cancers. The focus of research now turns to understanding the mechanisms by which ATX and LPA promote mammary tumourigenesis and metastasis. Targeting the ATX–LPA signalling axis for drug development may further improve outcomes in patients with breast cancer.

Mammary Tumorigenesis through LPA Receptor Signaling

Lysophosphatidic acid (LPA) is a lipid growth factor that is produced by an extracellular phospholipase, termed autotaxin (ATX), and acts via G protein-coupled receptors. In this issue of Cancer Cell, Liu et al. show that transgenic overexpression of ATX or LPA receptors leads to invasive and metastatic mammary cancer.

Expression of Autotaxin and Lysophosphatidic Acid Receptors Increases Mammary Tumorigenesis, Invasion, and Metastases

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Dermal Lymphatic Invasion and Inflammatory Breast Cancer Are Independent Predictors of Outcome After Postmastectomy Radiation

Inflammatory breast cancer (IBC) is a clinical staging based on history and physical findings. Dermal lymphatic invasion (DLI) can occur with or without IBC. We examine how these independently affect outcome in women treated with postmastectomy radiation. Four hundred thirty-two patients treated with postmastectomy radiation for invasive mammary cancer were assessed. Kaplan-Meier methodology was used to calculate rates of locoregional recurrence (LRR), distant metastases (DM) and overall survival (OS). Variables entered into univariate and multivariate analysis included T stage, IBC, DLI, estrogen receptor/progesterone receptor status, HER-2/neu status, N stage, extracapsular node extension (ECE), and use of chemotherapy (CT). Median follow-up is 58 months. For all 432 patients, the rate of LRR was 3% and DM 28%. Seven percent are alive with disease (AWD) and 26% are dead of disease (DOD). Thirty-one patients had IBC without DLI, 21 had DLI without IBC, and 18 had both IBC and DLI. For DLI 10% developed LRR, 45% DM, 7.5% are AWD and 50% are DOD. Of patients with IBC, 8% developed LRR, 44% DM, 8% are AWD and 48% DOD. DLI was the only significant independent predictor for LRR (HR 4.8, P < 0.05). Predictors of DM and OS were IBC, > or =4 positive nodes, and CT. DLI and IBC are independent predictors of poor outcome after postmastectomy radiation. DLI is associated with an increased risk for LRR, and IBC with worse rates of DM and OS. Patients with both features have worse outcome than those with either alone.

Detection of in situ mammary cancer in a transgenic mouse model: in vitro and in vivo MRI studies demonstrate histopathologic correlation

Improving the prevention and detection of preinvasive ductal carcinoma in situ (DCIS) is expected to lower both morbidity and mortality from breast cancer. Transgenic mouse models can be used as a ‘test bed’ to develop new imaging methods and to evaluate the efficacy of candidate preventive therapies. We hypothesized that despite its microscopic size, early murine mammary cancer, including DCIS, might be accurately detected by MRI. C3(1) SV40 TAg female mice (n = 23) between 10 and 18 weeks of age were selected for study. Eleven mice were subjected to in vitro imaging using a T2-weighted spin echo sequence and 12 mice were selected for in vivo imaging using a T1-weighted gradient echo, a T2-weighted spin echo and high spectral and spatial resolution imaging sequences. The imaged glands were carefully dissected, formalin fixed and paraffin embedded, and then H&E stained sections were obtained. The ratio of image-detected versus histologically detected cancers was obtained by reviewing the MR images and H&E sections independently and using histology as the gold standard. MR images were able to detect 12/12 intramammary lymph nodes, 1/1 relatively large (∼5 mm) tumor, 17/18 small (∼1 mm) tumors and 13/16 ducts distended with DCIS greater than 300 µm. Significantly, there were no false positives—i.e., image detection always corresponded to a histologically detectable cancer in this model. These results indicate that MR imaging can reliably detect both preinvasive in situ and early invasive mammary cancers in mice with high sensitivity. This technology is an important step toward the more effective use of non-invasive imaging in pre-clinical studies of breast cancer prevention, detection and treatment.

Cell proliferation of feline and human breast cancer cell types is inhibited by pomegranate juice

Mammary cancer, a devastating disease in both humans and companion animals, has been associated with numerous factors including diet. Polyphenolic antioxidants found in pomegranate fruits have been shown to reduce tumor burden and inhibit angiogenesis and cell growth.(Kim et al., 2002; Afaq et al., 2005; Malik et al., 2005) Feline mammary carcinomas (FMC) are known to have similar invasive behavior, histologic appearance, and overall poor prognosis to estrogen receptor negative (ER‐) invasive human mammary cancer.(Porrello et al., 2004; Zappulli et al., 2005) In this study, supplementation with an antioxidant‐rich whole food (pomegranate) was evaluated for anti‐cancer properties in an ER‐ human breast cancer cell line (MDA‐MB‐231) as a model for FMC. Antioxidant capacity and polyphenolic content of pomegranate juices (PJ) were characterized by reportable methods. ER‐ cells were exposed to PJ at various concentrations (0.5%, 1%, 5%) and to Cisplatin (5 ug/ml, 10 ug/ml, 15 ug/ml) doses for 48 and 72 h. MTT assays were performed to evaluate the ability of PJ to inhibit tumor cell growth. Statistical significance was determined using PROC GLM (SAS 9.1) with alpha = 0.05. Cell proliferation of the ER‐ cancer cells was inhibited by pomegranate juice in a dose‐ and time‐dependent manner (p &lt; 0.0001).Maximal inhibition was seen for pomegranate juice formulations at the 5% dose, and the response was comparable to that of high‐dose Cisplatin. This first phase study shows that PJ may be a useful nutrient‐based, non‐chemotherapeutic treatment alternative for the inhibition of ER‐ breast cancer cell proliferation.

Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERα-negative and ERα-positive mammary preneoplasia and cancer

BRCA1 can regulate estrogen receptor-alpha (ERalpha) activity. This study tested the hypotheses that Brca1 loss in mammary epithelium alters the estrogenic growth response and that exposure to increased estrogen or ERalpha collaborates with Brca1 deficiency to accelerate preneoplasia and cancer development. Longer ductal extension was found in mammary glands of Brca1(f/f;MMTV-Cre) mice during puberty as compared to wild-type mice. Terminal end bud differentiation was impaired in Brca1 mutant mice with preservation of prolactin-induced alveolar differentiation. Exogenous estrogen stimulated an abnormal sustained increase in mammary epithelial cell proliferation and the appearance of ERalpha-negative preneoplasia in postpubertal Brca1 mutant mice. Carcinogenesis was investigated using Brca1(f/f;MMTV-Cre) mice hemizygous for p53. Exogenous estrogen increased the percentage of mice with multiple hyperplastic alveolar nodules. Targeted conditional ERalpha overexpression in mammary epithelial cells of mice that were Brca1 mutant and hemizygous for p53 increased the percentage of mice exhibiting multiple hyperplastic nodules, invasive mammary cancers and cancer multiplicity. Significantly more than half of the preneoplasia and cancers were ERalpha negative even as their initiation was promoted by ERalpha overexpression.

Reflectance confocal microscopy for characterization of mammary ductal structures and development of neoplasia in genetically engineered mouse models of breast cancer

The earliest steps of breast cancer begin with aberrations in mammary ductal structure. Techniques that enable an investigator to image in situ and then analyze the same tissue using biochemical tools facilitates identification of genetic networks and signaling pathways active in the imaged structure. Cellular confocal microscopy (VivaCell-TiBa, Rochester, New York) is used to image mammary ductal structures and surrounding vasculature in situ in intact wild-type and genetically engineered mice that develop ER alpha-initiated ductal carcinoma in situ (DCIS) and ER alpha-driven invasive mammary cancer. In wild-type mice, normal mammary ductal structures that appear from puberty through lactation are visualized and serially sectioned optically, and a developmental atlas is created. Altering tissue preparation enabled visualization of the vasculature surrounding the ductal structures. In the genetically engineered mice, aberrant mammary ductal structures and cancers are imaged and compared to corresponding normal structures. Different preparation techniques are able to preserve tissue for routine histological analyses and RNA isolation. Comparative studies demonstrate that reflectance confocal imaging provides more cellular detail than carmine-alum-stained mammary gland whole mounts and equivalent detail with hematoxylin and eosin stained tissue sections. In summary, reflectance confocal microscopy is a tool that can be used to rapidly and accurately analyze mammary gland structure.

Prediction of the axillary lymph node status in mammary cancer on the basis of clinicopathological data and flow cytometry

Axillary lymph node status is a major prognostic factor in mammary carcinoma. It is clinically desirable to predict the axillary lymph node status from data from the mammary cancer specimen. In the study, the axillary lymph node status, routine histological parameters and flow-cytometric data were retrospectively obtained from 1139 specimens of invasive mammary cancer. The ten variables: age, tumour type, tumour grade, tumour size, skin infiltration, lymphangiosis carcinomatosa, pT4 category, percentage of tumour cells in G2/M- and S-phases of the cell cycle, and ploidy index were considered as predictor variables, and the single variable lymph node metastasis pN (0 for pN0, or 1 for pN1 or pN2) was used as an output variable. A stepwise logistic regression analysis, with the axillary lymph node as a dependent variable, was used for feature selection. Only lymphangiosis carcinomatosa and tumour size proved to be significant as independent predictor variables; the other variables were non-contributory. Three paradigms with supervised learning rules (multilayer perceptron, learning vector quantisation and support vector machines) were used for the purpose of prediction. If any of these paradigms was used with the information from all ten input variables, 73% of cases could be correctly predicted, with specificity ranging from 82 to 84% and sensitivity ranging from 60 to 63%. If only the two significant input variables were used, lymphangiosis carcinomatosa and tumour diameter, the prediction accuracy was no worse. Nearly identical results were obtained by two different techniques of cross-validation (leave-one-out against ten-fold cross validation). It was concluded that: artificial neural networks can be used for risk stratification on the basis of routine data in individual cases of mammary cancer; and lymphangiosis carcinomatosa and tumour size are independent predictors of axillary lymph node metastasis in mammary cancer.

Identification of Genes Associated with the Invasive Status of Human Mammary Carcinoma Cell Lines by Transcriptional Profiling

We derived the transcriptional profiles of four invasive and four noninvasive mammary carcinoma cell lines by Affymetrix GeneChip^® Technology with the profile of human mammary epithelial cells as a reference. We focused on the identification of genes which are upregulated in the invasive cell lines based on the following threshold levels: -fold change of 2 or higher in at least three or more cell lines. According to the scoring criteria as described above, we identified 18 transmembrane receptors, 18 secreted proteins and 5 kinases. Several of the genes described have already been put into context with respect to invasion of mammary carcinoma. We therefore focused on deregulated genes for which such an association has not been described before: transmembrane receptor tyrosine kinase DDR2, transmembrane receptors PMP22 and EMP3 and cell adhesion molecule N-cadherin. Making use of real-time PCR, consistently increased steady-state levels of mRNAs for these genes were found in an extended panel of invasive and noninvasive mammary cancer cell lines.

Significance of metallothionein expression in breast myoepithelial cells

Metallothioneins (MTs), a group of cysteine-rich proteins with a small molecular mass, are known to have metalloregulatory functions. MT gene expression has been demonstrated to be cell type-specific and differentially regulated (possibly related to their germ layer origin and different functional states). In vitro studies suggest that MT-2A, MT-IE, and MT-1F isoforms may be related to breast cancer. In this study, data on MT-2A, MT-1E, MT-1F mRNA analysis via reverse transcription-polymerase chain reaction in invasive ductal breast cancer tissues and their adjacent benign breast tissues from 27 mastectomies are presented. Expression of mRNA in all the three MT isoforms was detected in both cancerous and adjacent benign breast tissues (with MT-2A mRNA expression being the highest). MT-1F expression was significantly higher in benign breast tissues compared with the breast cancers (P=0.017). In situ hybridization confirmed the expression of MT-2A mRNA in the myoepithelial cells of the breast tissues. Immunohistochemical localization of the MT protein revealed that myoepithelial cells consistently expressed the MT protein, while the cancer cells expressed MT with great variation. Based on our immunohistochemical and mRNA analysis, it is likely that the three MT isoforms are specifically expressed in myoepithelial cells of benign breast tissues and cancer cells of the invasive ductal breast cancer tissues. As MT expression occurs in myoepithelial cells and ductal breast cancer cells, our finding supports the proposition that loss of myoepithelial cells in invasive mammary cancers may be compensated in part by changes in the tumor cells, which may subsequently be the basis for studying the role of MT in breast physiology and carcinogenesis. Differential MT-1F expression in breast myoepithelial cells warrants further study.