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Background: The cytomorphological features in the distinction between phyllodes tumour (PT) and fibroadenoma (FA) on fine needle aspiration biopsy (FNAB) remains challenging because of the biphasic nature of these lesions and the rarity of PT., Methods: FNAB smears of histologically confirmed PT (N = 26) and FA (N = 78) cases were retrieved from a retrospective database interrogation from the Department of Cytology/Tissue Pathology, ICPMR Pathology West (Cerner Millennium) in Westmead Hospital. For each case, two smears were selected, de-identified and independently reviewed by four observers comprising two cytologists and two cytopathologists. Cytological parameters examined included detailed evaluation of smear cellularity, epithelial and stromal components as well as the smear background., Results: The cytological features of moderate to marked stromal cellularity and stromal nuclear atypia were more evident in PT than in FA, identified by three out of four observers. The epithelial characteristics, presence of macrophages, multinucleated giant cells and blood vessels showed no statistically significant differences in the distinction between the two lesions., Conclusion: The results of this study indicate that in all of the cytological features assessed for PT and FA, no single cytological feature was found to be statistically significant in identifying PT across all observers. This reflects the overlap of cytological features seen in these lesions. FNAB cytology cannot reliably distinguish FA and PT., (© 2018 Wiley Periodicals, Inc.)

Measurement of endogenous free and bound NAD(P)H relative concentrations in living cells isa useful method for monitoring aspects of cellular metabolism, because the NADH∕NAD⁺ reduction-oxidation pair is crucial for electron transfer through the mitochondrial electron transport chain. Variations of free and bound NAD(P)H ratio are also implicated in cellular bioenergetic and biosynthetic metabolic changes accompanying cancer. This study uses two-photon fluorescence lifetime imaging microscopy (FLIM) to investigate metabolic changes in MCF10A premalignant breast cancer cells treated with a range of glycolysis inhibitors: namely, 2 deoxy-D-glucose, oxythiamine, lonidamine, and 4-(chloromethyl) benzoyl chloride, as well as the mitochondrial membrane uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Through systematic analysis of FLIM data from control and treated cancer cells, we observed that all glycolytic inhibitors apart from lonidamine had a slightly decreased metabolic rate and that the presence of serum in the culture medium generally marginally protected cells from the effect of inhibitors. Direct production of glycolytic L-lactate was also measured in both treated and control cells. The combination of these two techniques gave valuable insights into cell metabolism and indicated that FLIM was more sensitive than traditional biochemical methods, as it directly measured metabolic changes within cells as compared to quantification of lactate secreted by metabolically active cells.

Background: The WHI trial suggests an increase of breast cancer in postmenopausal women probably according to the progestogenic compound, i.e. medroxyprogesterone acetate (MPA). However, the mechanism for a possible carcinogenic effect of MPA remains unclear so far. Progesterone receptor membrane component-1 (PGRMC1) may be important in tumorigenesis and thus may increase breast cancer risk. We investigated the influence of MPA alone and in combination with growth factors on breast cancer cells overexpressing PGRMC1., Methods: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12 cells). Cells transfected only with the vector were used as control cells (EVC-cells). Medroxyprogesterone acetate (MPA), norethisterone (NET) and progesterone (P) were tested alone and in combination with a mixture of growth factors. Cell proliferation was measured by MTT assay., Results: The growth factor mixture (GF) was able to induce cell proliferation in both cell types, however, the effect was much higher in the WT-12 cells. In WT-12 cells both MPA and NET alone significantly increased cell proliferation with values of 40% and 97%, respectively. Progesterone, however, had no effect. In combination with GF MPA significantly further enhanced cell proliferation as compared to the effect of MPA alone and GF alone in both cell lines. NET showed no further increase as compared to NET alone and P had no effect., Conclusions: We could demonstrate a significant proliferative effect of MPA when combined with high concentrations of growth factors. This effect was more pronounced in breast cancer cells overexpressing PGRMC1. These results may be of clinical relevance since in the combined WHI trial an increased breast cancer risk was found during treatment with conjugated equine estrogens plus MPA., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: Clinical trials have demonstrated an increased risk of breast cancer during estrogen/norethisterone (NET) therapy. With this in mind, the effects of estrogen/NET combination on the proliferation of breast cancer cells overexpressing the progesterone receptor membrane component 1 (PGRMC1) were examined. The same combination was used for the first time in a mouse xenograft model to determine its effects on tumor development., Methods: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12 cells) or empty vector control (pcDNA-3HA). NET, medroxyprogesterone acetate (MPA), and progesterone were tested alone and sequentially and continuously combined with estradiol (E2). Six-week-old nude mice were inoculated with E2 pellets 24 hours before the injection of tumor cells into both flanks (n = 5-6 mice per group). After 8 days, animals were inoculated with a NET pellet or with placebo pellets, and tumor volumes were recorded twice a week., Results: NET alone significantly increased the proliferation of WT-12 cells, MPA was effective only at the two highest concentrations, and progesterone had no effect. The twofold to threefold E2-induced increase (10 M) was not significantly influenced by the addition of the various progestogens. In contrast, 10 M E2 had no effect; however, addition of MPA and NET triggered a significant proliferative response. In vivo, a sequential combination of NET and E2 also significantly increased the tumor growth of WT-12 cells; empty vector cells did not respond to NET., Conclusions: We have demonstrated for the first time that an E2/NET combination increases the proliferation of PGRMC1-overexpressing breast cancer cells, both in vivo and in vitro. Our results suggest that undetected tumor cells overexpressing PGRMC1 may be more likely to develop into frank tumor cells in women undergoing E2/NET hormone therapy.

Objectives: Dienogest (DNG) is already used in hormone therapy, since recently being also the progestogenic component of the first estradiol based contraceptive pill. Data on breast cancer risk are currently not available. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in tissues of breast cancer patients and has already been proposed as a predictor for breast cancer risk., Methods: MCF-7 cells overexpressing PGRMC1 were stimulated with DNG, medroxyprogesterone acetate (MPA), norethisterone (NET) and progesterone (P) as well as sequentially and continuously combined with estradiol (E2)., Results: DNG and MPA alone elicited a significant proliferation at 10⁻⁶ and 10⁻⁵ M. NET increased cell proliferation at all concentrations tested whereas P showed no effect. E2 alone elicited a significant increase at 10⁻¹⁰ M, no effect was seen at 10⁻¹² M. Addition of the progestins (10⁻⁶ M) to E2 at 10⁻¹⁰ M had, compared to E2 only, no additional proliferating effect. However, at the low E2 concentration, DNG, MPA and NET significantly increased the E2-stimulated cell proliferation., Conclusion: DNG increased proliferation alone and in combination with low E2 concentrations. Thus a progestogen-derived breast cancer risk in the presence of low E2 concentrations cannot be excluded at least in women overexpressing PGRMC1.

Objectives: Recently the first monophasic contraceptive pill containing estradiol has been developed which is thought to be a milestone in contraception. Nomegestrol acetate (NOM) is the progestogenic component. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the tissue of breast cancer patients, and can predict a progestogen dependent risk of breast cancer., Methods: MCF-7 cells were transfected with PGRMC1 expression plasmid, and were stimulated with estradiol (E2, 10(-12) and 10(-10) M). NOM, progesterone (P), medroxyprogesterone acetate (MPA) and norethisterone (NET) (each 10(-7) M) were added sequentially or continuously., Results: E2 at 10(-10) M elicited a significant increase of cell proliferation from 150 to 200%. No effect was seen at 10(-12) M. Addition of the progestogens to E2 at 10(-10) M had no significant effect. However, at an E2 10(-12) M, NET significantly stimulated cell proliferation more pronounced in the continuous combined model. No effect was seen for NOM, P and MPA. The E2/NET combined effect could be abrogated by the addition of an estrogen receptor (ER) antagonist., Conclusion: Since NOM did not increase proliferation it may be concluded that it will be neutral in terms of breast cancer risk when combined with E2 at least in women overexpressing PGRMC1.

Objectives: Evidence is accumulating that progestogens may play a crucial role in the development of breast cancer under contraception and hormone therapy in reproductive and menopausal women. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of progesterone and nine synthetic progestins on MCF-7 breast cancer cells overexpressing PGRMC1., Methods: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12). To test the effects of progestogerone (P) and the synthetic progestins chlormadinone acetate (CMA), desogestrel (DSG), drospirenone (DRSP), dydrogesterone (DYD), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), nomegestrol (NOM) and norethisterone (NET) on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations (0.01-1 µmol/l)., Results: In MCF-7 cells, DRSP, DSG, DYD, LNG and NET increased the proliferation at 1 µmol/l, the effect being highest for NET with about 20%. In WT-12 cells, the same progestins, but additionally MPA, showed a significant increase, which was much higher (30-245%) than in MCF-7 cells. Here again, NET showed the highest proliferative effect. No effect was found for CMA, NOM and P., Conclusion: Some synthetic progestins trigger a proliferative response of PGRMC1-overexpressed MCF-7 cancer cells. The effect of progestogens on breast cancer tumorigenesis may clearly depend on the specific pharmacology of the various synthetic progestins.

Objective: Breast cancer risk is still an important topic regarding hormone therapy as well as oral contraception. Evidence that progestogens may play a crucial role is accumulating. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of different estradiol (E2) concentrations and the addition of two progestogens on MCF-7 breast cancer cells overexpressing PGRMC1., Methods: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (MCF-7/PGRMC1-3HA [WT-12]). To test the effects of E2 and progestogens on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations of E2 (10 and 10 M) alone and in combination with progesterone and medroxyprogesterone acetate (each 10 M)., Results: E2 elicited a concentration-dependent proliferative effect on both cell lines, which was much more pronounced in WT-12 cells (50% vs 200%). This effect could be completely abrogated by the addition of the E2 antagonist fulvestrant. Addition of progesterone had no influence on the E2-induced effect, whereas medroxy-progesterone acetate enhanced the E2-induced effect at a low E2 concentration, which was, again, more pronounced in the WT-12 cells. The figures were between 20% and 40% in MCF-7 and between 60% and 250% in WT-12 cells., Conclusions: Overexpression of PGRMC1 sensitizes the proliferative response of the MCF-7 breast cancer cell line to estradiol. The effect of progestogens on breast cancer tumorigenesis may depend on the specific progestogen used for hormone therapy or oral contraception.

Objective: Progesterone influences mammary gland development and probably breast cancer tumorigenesis and functions by regulating a broad spectrum of physiological processes. We investigated receptor membrane-initiated actions of progesterone in MCF-7 breast cancer cells via progesterone receptor membrane component 1 (PGRMC1)., Design and Method: The expression of PGRMC1 in breast cancer was verified by immune fluorescent analysis of paraffin sections. MCF-7 cells were transfected with PGRMC1 (wild type) or PGRMC1 variants. These cells were stimulated with a membrane-impermeable progesterone (P4) conjugate (P4-BSA-fluorescein isothiocyanate, P4-BSA-FITC, 10(-6) mol/l) or unconjugated progesterone (P4, 10(-6) mol/l) in the presence or absence of the progesterone receptor blocker RU-486 (10(-6) mol/l). Additionally, the effects on the expression of vascular endothelial growth factor A (VEGF-A) were determined using quantitative real-time polymerase chain reaction., Results: PGRMC1 is perinuclearly localized in breast cancer cells. Western Blot analysis suggests that PGRMC1 is phosphorylated at serine 180. MCF-7-PGRMC1 (S180A) cells show an approximately 35% increase in proliferation after incubation with P4-BSA-FITC compared to MCF-7 control and MCF-7-PGRMC1 (wild type) cells. This effect cannot be blocked by RU-486. P4 reduced proliferation of MCF-7-PGRMC1 cells by approximately 10% compared to untreated controls. P4-BSA-FITC treatment led to a roughly three-fold activation of VEGF-A gene expression compared to MCF-7 cells., Conclusion: PGRMC1 is expressed in breast cancer tissue and mediates an RU-486-independent proliferative signal. It might also contribute to VEGF-induced neovascularization in tumor tissue. Thus, screening for PGRMC1 expression might be of interest to identify women with a higher expression of PGRMC1 and who might thus be susceptible for breast cancer development under hormone replacement therapy.

Introduction: Breast tumors lacking the estrogen receptor-alpha (ER-alpha) have increased incidence of resistance to therapy and poorer clinical prognosis., Methods: Whole tissue sections from 16 cryopreserved breast cancer tumors that were either positive or negative for the ER (eight ER positive and eight ER negative) were differentially analyzed by multiplex imaging of two-dimensional PAGE gels using 54 cm isoelectric focusing. Differentially detected spots of Progesterone Receptor Membrane Component 1 (PGRMC1) were shown to differ in phosphorylation status by differential two dimensional polyacrylamide gel electrophoresis of phosphatase-treated tumor proteins. Site directed mutagenesis was used to create putative phosphorylation site point mutants in PGRMC1. Stable transfectants of these mutants in MCF7 cells were assayed for their survival after oxidative stress, and for AKT kinase phosphorylation. Immune fluorescence using anti-PGRMC1 monoclonal antibody 5G7 was performed on breast cancer tissue microarrays., Results: Proteins significantly differentially abundant between estrogen receptor negative and estrogen receptor positive tumors at the 0.1% level were consistent with published profiles, suggesting an altered keratin pool, and increased inflammation and wound responses in estrogen receptor negative tumors. Two of three spots of PGRMC1 were more abundant in estrogen receptor negative tumors. Phosphatase treatment of breast tumor proteins indicated that the PGRMC1 isoforms differed in their phosphorylation status. Simultaneous mutation of PGRMC1 serine-56 and serine-180 [corrected] fully abrogated the sensitivity of stably transfected MCF7 breast cancer cells to peroxide-induced cell death. Immune fluorescence revealed that PGRMC1 was primarily expressed in ER-negative basal epithelial cells of mammary ductules. Even in advanced tumors, high levels of ER or PGRMC1 were almost mutually exclusive in individual cells. In five out of five examined ductal in situ breast cancers of comedo type, PGRMC1 was expressed in glucose transporter 1 negative or positive poorly oxygenated cells surrounding the necrotic core, surrounded by a more distal halo of ER-positive cells., Conclusions: PGRMC1 phosphorylation may be involved in the clinical differences that underpin breast tumors of differing ER status.

Breast cancer is one of the most common cancers observed in women in industrialized Western countries. The development of novel diagnostic methods and the application of modern systemic therapies have significantly optimized early detection and therapy of breast cancer. However, many patients are currently overtreated. Traditionally, tumours have been categorized on the basis of histopathological criteria. However, staining pattern and intensity of cancer cells are not sufficient to reflect the molecular events driving tumour development and progression. Therefore, new genomic, transcriptomic and proteomic techniques are applied to clinical samples aiming to identify new targets for a therapy tailored for an individual patient. After an introduction to common genomic and transcriptomic profiling technologies and their relevance for clinical use, we will focus on analytical and preanalytical applications for the identification of new therapeutic targets by protein profiling, with a special emphasis on two-dimensional gel-technologies (2D-PAGE), particularly as they apply to the study of breast cancer.

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.