Ethinylestradiol is beneficial for postmenopausal patients with heavily pre-treated metastatic breast cancer after prior aromatase inhibitor treatment: a prospective study.

BACKGROUND: Oestrogens usually stimulate the progression of oestrogen receptor (ER)-positive breast cancer. Paradoxically, high-dose oestrogens suppress the growth of these tumours in certain circumstances. METHODS: We prospectively examined the efficacy and safety of ethinylestradiol treatment (3 mg per day oral) in postmenopausal patients with advanced or recurrent ER-positive breast cancer who had previously received endocrine therapies, especially those with resistance to aromatase inhibitors. RESULTS: Eighteen patients were enrolled with the median age of 63 years and the mean observation time of 9.2 months. Three cases withdrew within 1 week due to oestrogen flare reactions with nausea, fatigue and muscle-skeletal pain. The response rate was 50% (9 out of 18), and the clinical benefit rate was 56% (10 out of 18). The stable disease (<6 months) was 17% (3 out of 18) and another 2 cases were judged as progressive disease. Time-to-treatment failure including 2 on treatment was a median of 5.6 months (range 0.1 to 14.5(+)). Although vaginal bleeding or endometrial thickening was observed in patients receiving long-term treatment, there were no severe adverse events, such as deep venous thrombosis or other malignancies. CONCLUSION: Although the mechanism of this treatment has not been fully understood, our data may contribute to change the common view of late-stage endocrine therapy.

Generation of ERα-floxed and knockout mice using the Cre/LoxP system.

Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

[Lung cancer detected by fluorodeoxyglucose-positron emission tomography/computed tomography in the course of Mycobacterium avium infection; report of a case].

The patient was 64-year-old male. Chest computed tomography (CT) scan revealed an 18 mm of nodular lesion in the right upper lobe, in which inflammatory lesions due to the Mycobacterium avium infection was preexisted. On fluorodeoxyglucose-positron emission tomography (FDG-PET)/CT scan, value of standard uptake value (SUV) max was 4.0. This finding may be caused by the inflammatory change but the malignancy was more likely with a concomitant finding of elevated serum carcinoembryonic antigen (CEA). Surgical resection by right upper lobectomy was performed. Postoperative pathology confirmed the existence of adenocarcinoma in the lesions of epithelioid granuloma with giant cells. FDG-PET/CT contributed effectively to detect a malignancy in the inflammatory lesions of Mycobacterium avium infection.

IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic disease with a Th2-type-cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half-life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)-4/IL-13 antagonist (IL-4DM), which blocks both IL-4 and IL-13 signal transductions. OBJECTIVE: To examine the effects of IL-4DM in vivo in an AD model induced by the repeated exhibition of oxazolone (OX). METHODS: Plasmid DNA was injected intraperitoneally to cause an experimental AD-like dermatitis. The effect was evaluated by ear thickness, histological findings, and mast cells counts in the inflamed skin. The plasma IgE and histamine levels were measured. Cytokine production in skin and splenocytes were also analysed. RESULTS: Mice treated with control plasmid developed marked dermatitis with mast cells and eosinophil infiltration, and had increased plasma IgE and histamine levels with a Th2 type splenocyte cytokine profile. Treatment with mouse IL-4 DNA augmented the ear swelling and thickness with an increased dermal eosinophil count, plasma histamine level, and production of splenocyte IL-4. However, IL-4DM treatment successfully controlled the dermatitis, decreased the mast cell and eosinophil count, and suppressed plasma IgE and histamine levels. Splenocytes produced an increased level of IFN-gamma. CONCLUSION: These data showed that the simultaneous suppression of IL-4/IL-13 signals successfully controlled Th2-type chronic dermatitis. IL-4DM DNA treatment is a potent therapy for AD and related diseases.

Sexual differentiation in sensitivity to male body odor(1).

We have confirmed that more female subjects than male subjects evaluate male body odor as significantly unpleasant. Through an investigation on sexual differentiation in sensitivity to male body odor, we concluded that one of the volatile steroids, androstenone, had two effects on female olfactory sense. First, female subjects perceived androstenone itself to be more unpleasant than male subjects. Second, for only female subjects, androstenone, at a concentration of one-tenth of detection threshold, enhanced the intensity and unpleasantness of body-odor constituents such as short-chain fatty acids.

Transcription factor EGR3 is involved in the estrogen-signaling pathway in breast cancer cells.

Estrogen has been closely associated with the genesis and malignant progression of breast cancer. However, the molecular mechanism underlying the effects of estrogen is far from being completely clarified. We previously developed a custom-made cDNA microarray consisting of approximately 200 estrogen-responsive genes in breast cancer cells. Using this system, we found one estrogen-induced gene in various cancer cell lines, including breast cancer MCF-7 cells, which encode a zinc-finger transcription factor, EGR3 (early growth response 3). Northern blot analysis of estradiol-treated MCF-7 cells showed rapid and robust induction of Egr3, and addition of cycloheximide or ICI 182,780 suggested that Egr3 is the bona fide target for the estrogen receptor alpha (ERalpha). Using stable transformants derived from MCF-7 cells which were transfected with expression-controllable Egr3-expression vector, we demonstrated that Nab2 is one of the target genes for EGR3. Microarray analysis of the transformants revealed other candidate EGR3-induced genes. These strategies could be useful for analyzing downstream genes of ERalpha, and may contribute to elucidating the extensive signaling network of estrogen stimuli. Furthermore, a reporter assay using the upstream region of fasL probably involving escape from the immune system revealed that fasL is another target gene for EGR3. The roles of EGR3 in the physiology of breast cancer are discussed.

Clinical significance of AIB1 expression in human breast cancer.

AIB1 (amplified in breast cancer 1) is a member of the steroid receptor coactivator family and is a key factor in enhancing estrogen-dependent transcription. To evaluate the clinical significance of AIB1 in breast cancer, we performed Southern blot analysis of the AIB1 gene on 124 human breast cancer tissues. We also performed reverse transcription-polymerase chain reaction and semi-quantitative analysis of AIB1 mRNA expression on 58 of the tissues, and immunohistochemical detection of AIB1 protein on 115 of the tissues. On Southern blot analysis, the AIB1 gene was amplified in only two of the 124 breast cancer cases. On semi-quantitative analysis, the relative expression level of AIB1 normalized to that of GAPDH varied from 0.247 to 7.721 (median = 0.94), and was not correlated with any clinico-pathological factors. Although most of the breast cancer cells revealed cytoplasmic staining of AIB1, only 16% (18 in 115) showed nuclear staining of AIB1 protein. AIB1 nuclear expression was correlated with positivity for estrogen receptor alpha (P = 0.022). Those patients with tumor samples that showed nuclear staining of AIB1 tended to be successfully treated by endocrine therapy in comparison with those who did not show nuclear staining of AIB1. In conclusion, AIB1 nuclear expression was correlated with the estrogen receptor alpha status, and patients with AIB1 nuclear expression tended to be successfully treated by hormonal therapy.

The expression and function of estrogen receptor alpha and beta in human breast cancer and its clinical application.

The overexpression of estrogen receptor alpha (ERalpha) is frequently observed in the early stage of breast cancer. We previously reported that the specific promoter of the ERalpha gene is responsible for this enhanced transcription of the gene, and identified the cis-acting elements which play an important role in its transcription. Furthermore, methylation of the ERalpha gene promoters also contribute to the regulation of gene transcription. Elucidation of these mechanisms of ERalpha gene expression may provide useful information for the early detection and chemoprevention of breast cancer. On the other hand, the expression of ERbeta has been reported in breast cancer. We have also assessed the significance and function of ERbeta and its variant types in breast cancer, and suggest that ERbeta and ERbetacx specifically suppress the function of ERalpha through different mechanisms. ERbeta isoforms may be important functional modulators of the estrogen-signaling pathway in breast cancer cells, and might affect the clinical outcome of patients. Moreover, to address the role of these ERs on the estrogen-dependent growth of breast cancer cells and to develop a diagnostic tool, we have analyzed the gene expression profiles of estrogen-responsive genes using cDNA microarray. Based on these results, the expression of several candidate genes in breast cancer tissues were analyzed by real-time RT-PCR and by immunohistochemical techniques, in order to discover new predictive factors for the endocrine therapy of patients with breast cancer. These studies could provide new clues for the elucidation of the estrogen-dependent mechanisms of cancer and the clinical benefits for patients.

Development of cDNA microarray for expression profiling of estrogen-responsive genes.

Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed.

Evaluation of oestrogen receptor beta wild-type and variant protein expression, and relationship with clinicopathological factors in breast cancers.

We addressed the clinicopathological significance of the oestrogen receptor (ER) beta protein, including an ERbeta variant, ERbetacx, in normal human breast and breast cancer. The reverse transcriptase-polymerase chain reaction (RT-PCR) showed that wild-type ERbeta (ERbetaw) mRNA expression was higher in normal than cancer tissues, and that ERbetacx mRNA was higher in cancer than in normal tissues. Immunohistochemistry of 22 normal breast tissues and 57 breast cancers was performed with three different ERbeta antibodies and one ERbetacx antibody. All normal breast samples showed staining with the three ERbeta antibodies, suggesting that ERbetaw might have a physiological role in oestrogen signalling in the normal breast. In breast cancer, expression of the ERbetaw protein correlated well with the expression of the ERalpha and progesterone receptor (PgR), as well as histological grade (HG), and tended to indicate a better prognosis than when ERbetaw was absent. Thirty-one (54%) breast cancer samples contained ERbetacx, whereas the corresponding tissue for normal breast samples stained positive in only two (9%).

Expression of fas/fas ligand (fasL) and its involvement in infiltrating lymphocytes in hepatocellular carcinoma (HCC).

BACKGROUND: This study was conducted to examine the expression of Fas/Fas ligand (FasL), to elucidate its relationship with tumor-infiltrating lymphocytes (TILs), and to detect possible gene mutation of Fas/FasL in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODS: Indirect immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of liver biopsy and surgery specimens from five normal livers, and from the livers of 30 patients with HCC. Fas/FasL mRNA-expressing cells and apoptotic cells were detected by in situ hybridization and DNA nick end labeling (TUNEL), respectively. We also performed polymerase chain reaction (PCR)-amplifying and direct sequencing for the Fas/FasL gene. RESULTS: Fas/FasL and its mRNA were localized on the membrane or in the cytoplasm in some HCC cells, as well as hepatocytes. Their expression was enhanced in areas with infiltrating inflammatory cells in the noncancerous regions of liver tissue and on the margins of the cancerous tissue. The positivity rate for TUNEL was elevated along these margins. The labeling index of Fas/FasL was lower in the cancerous liver tissue than in the surrounding noncancerous region (P < 0.01), and tended to decrease in proportion to the malignancy of tumor cells; Fas/FasL expression was not found on poorly differentiated type cancer cells. Fas(-)/FasL(+), FasL-mRNA(+) HCC cells were seen in one specimen of moderately differentiated type. Some CD8+T lymphocytes were TUNEL-positive around the cancerous region. In this study, cancerous and noncancerous tissues in HCC revealed no genetic mutations in any exons of Fas/FasL. CONCLUSIONS: These findings suggest that Fas/FasL expression was decreased in proportion to the malignancy of tumor cells, and that infiltrating CD8+ T lymphocytes play a role in apoptosis in HCC. The apoptosis in HCC could be regulated by the suppression of Fas/FasL expression, or, sometimes, by the enhancement of FasL expression.

Expression, function, and clinical implications of the estrogen receptor beta in human lung cancers.

The higher frequency of human lung adenocarcinoma in females than in males, strongly suggests the involvement of gender dependent factors in the etiology of this disease. This is the first investigation of estrogen receptor (ER) beta in human lung. Immunohistochemical staining revealed ERbeta expression in normal lung and in atypical adenomatous hyperplasia (AAH), considered as a precancerous lesion for adenocarcinomas. Adenocarcinomas showed significantly higher expression of ERbeta than squamous cell carcinomas. On the contrary, ERalpha expression was not detected in all cases. The functional integrity of ERbeta such as the binding ability to estrogen responsive element (ERE) and transcriptional activity was confirmed using a human lung cancer cell line, RERF-LC-OK. Colony formation of this cell was significantly reduced in the presence of pure antiestrogen. We conclude that ERbeta, but not ERalpha, is present in lung tissues with an important physiological function in normal lung. Furthermore, ERbeta may play a role in growth and development of adenocarcinomas.

Increased matrix metalloproteinase 9 activity and mRNA expression in lung ischemia-reperfusion injury.

OBJECTIVES: In lung ischemia-reperfusion injury, neutrophil migration from the vasculature to the interstitial spaces plays a major role in tissue injury. Degradation of the basement membrane, which is composed of extracellular matrix (ECM) molecules, is necessary for neutrophil migration. Matrix metalloproteinases (MMPs) might play a role in ECM degradation in lung ischemia-reperfusion injury. We evaluated the changes in the activity of MMP-2 and MMP-9, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expressions using rat lung transplantation models. METHODS: We divided animals into 4 groups. Groups I and II served as control groups with intact lungs (Group I) and 24-hour cold-preserved lungs (Group II). Groups III and IV received lung grafts after 24-hour cold preservation. The recipient animals were sacrificed 1 hour (Group III) or 24 hours (Group IV) after transplantation. We evaluated lung injury histologically. We assessed MMP activity using zymography. We assessed MMP-2, MMP-9, and TIMP-1 gene expression using biplex reverse transcriptase-polymerase chain reaction method. RESULTS: In Groups III and IV, we noted severe ischemia-reperfusion injury. We noted no significant difference in enzyme activity and gene expression of MMP-2 between Groups I and IV. The MMP-9 activity and gene expression were low during ischemia and increased on reperfusion. TIMP-1 gene expression was low during ischemia and at the early phase of reperfusion, and showed a dramatic increase at the late phase of reperfusion. CONCLUSIONS: Matrix metalloproteinase 9, but not MMP-2, may play an important role in ischemia-reperfusion injury. TIMP-1 increases at the late phase of reperfusion and may compensate for the activity of MMP-9.

Immunohistochemical analysis on biological markers in ductal carcinoma in situ of the breast.

BACKGROUND: The increasing use of mammographic screening has led to an increased detection of ductal carcinoma in situ (DCIS) of the breast. The detailed biological characteristics of DCIS and a new classification of DCIS based on these characteristics are needed. METHODS: Immunohistochemical studies were performed to assess the expression of c-erbB-2 (ErbB-2), estrogen receptor (ER), p53 and proliferative activity (Ki-67) in 65 patients with pure DCIS and 60 with invasive ductal carcinoma (IDC). We classified pure DCIS tumors using three classifications, the architectural, Nottingham, and Van Nuys classifications. RESULTS: ErbB-2, ER and p53 staining was positive in 34%, 66% and 21% of patients with DCIS, respectively, and 58%, 42% and 33% in patients with IDC, respectively. Ki-67 stained positively in 1.5% of patients with DCIS and 11.2% of patients with IDC. The comedo type showed a high rate of positive ErbB-2 and p53 staining. The cribriform and papillary types showed a high rate of positive ER staining. Under the Van Nuys classification, ErbB-2, p53 and Ki-67 expression were highest in the group with high nuclear grade and lowest in the group with non-high nuclear grade without necrosis. CONCLUSION: Although the biological markers of IDC tended to suggest aggressive behavior more so than those of DCIS, these differences were based on the histological sub-type, comedo or non-comedo. The Van Nuys classification best defined the subgroups of DCIS with a distinct expression pattern of biological markers, and the best candidates for breast-conserving surgery.

Localization of gelatinolytic activity can be detected in breast cancer tissues by film in situ zymography.

BACKGROUND: Extracellular matrix-degrading proteinases secreted by malignant tumor cells have been thought to play an essential role in the processes of invasion and metastasis. However, existence and localization of gelatinase activity in breast cancer tissues have not been clarified. METHODS: We developed a novel film for highly reproducible detection and the localization of gelatinolytic activity. This film has a gelatin layer with a constant thickness 7 microm, and adequate crosslinking to control the speed of degradation by proteases. Cryosections of several breast cancer tissues were put on this gelatin film and incubated for 16 hrs at 37 degrees C. After staining with ponceau 3R dye, the digested area was evaluated under light microscopy. RESULTS: Digestion of gelatin was detected in more than 90%of breast cancer specimens, although it varied in degree and area for each case. In most cases, the gelatinolytic activity was located within cancer nests, and was not detected in stromal cells surrounding cancer cells. The gelatinolytic activity was inhibited by 1,10-phenanthroline, an inhibitor of matrix metalloproteinases (MMPs). CONCLUSIONS: In this study, the localization of net MMP activity was confirmed in breast cancer nest using film in situ zymography. Detailed analysis on the relationship between the strength or distribution of MMP activity and malignancy are anticipated in the future.

Telomerase activity levels for evaluating the surgical margin in breast-conserving surgery.

To evaluate the efficiency of measuring telomerase activity levels in clinical diagnosis, we performed a semiquantitative analysis of telomerase activity in breast tumors and compared the results with the histological findings. Breast tissue adjacent to areas of cancer were also serially resected and checked for telomerase activity. The amount of telomerase activity in the breast cancers ranged widely, from 0.36 to 1180 units/microg, with 31 of the 34 (91.2%) showing a value above 1.0unit/microg. None of the normal breast tissues including mastopathy, and only 4 (23.5%) of 17 benign breast masses had values above 1.0unit/microg. Telomerase activity was detectable in serial sections of adjacent tissues as far as 10mm from the macroscopic tumor margin with histologically detectable cancer cells. Furthermore, telomerase activity was detectable in the scrape specimens obtained from the stump of the surgical margins for breast-conserving surgery, and this activity was in accordance with the histological findings. These findings show that conducting a semiquantitative assay of telomerase activity is useful for evaluating the surgical margin in breast-conserving surgery.