Yuichi Iino

Zoledronic Acid Significantly Reduces Skeletal Complications Compared With Placebo in Japanese Women With Bone Metastases From Breast Cancer: A Randomized, Placebo-Controlled Trial

PURPOSE To investigate the efficacy and safety of zoledronic acid for the treatment of bone metastases from breast cancer. PATIENTS AND METHODS Women with bone metastases (N = 228) were randomly assigned to receive 4 mg zoledronic acid (n = 114) or placebo (n = 114) via 15-minute infusions every 4 weeks for 1 year. The primary efficacy end point was the skeletal-related event (SRE) rate ratio between treatment groups. An SRE was defined as pathologic fracture, spinal cord compression, and radiation or surgery to bone. Secondary end points included percentage of patients with at least one SRE, time-to-first SRE, and Andersen-Gill multiple-event analysis. RESULTS The SRE rate ratio at 1 year (excluding HCM and adjusted for prior fracture) was 0.61 (permutation test; P = .027), indicating that zoledronic acid reduced the rate of SRE by 39% compared with placebo. The percentage of patients with at least one SRE (excluding HCM) was significantly reduced by 20% by zoledronic acid (29.8% v 49.6% for placebo; P = .003). Zoledronic acid significantly delayed time-to-first SRE (median not reached v 364 days; Cox regression; P = .007) and reduced the risk of SREs by 41% in multiple event analysis (risk ratio = 0.59; P = .019) compared with placebo. Zoledronic acid was well tolerated with a safety profile similar to placebo. No patient treated with zoledronic acid had grade 3 or 4 serum creatinine increase. CONCLUSION Zoledronic acid significantly reduced skeletal complications compared with placebo across multiple end points in Japanese women with bone metastases from breast cancer.

Immunohistochemical Expression of Metallothionein in Invasive Breast Cancer in Relation to Proliferative Activity, Histology and Prognosis

Immunohistochemically detected metallothionein expression [MT(+)] was shown to be related to aggressive behavior of the invasive ductal carcinoma of the breast. In this study, MT expression was examined immunohistochemically in 92 cases of invasive breast carcinoma and compared with immunohistochemically demonstrated estrogen receptor (ER), c-erbB-2, Ki-67 status and clinicopathological characteristics. Of the 92 cases examined, 27.1% (25 cases) were MT(+), and high percentages of the solid tubular subtype of invasive ductal carcinoma (47%), medullary carcinoma (80%), and carcinomas with spindle cell metaplasia (100%) were positive for MT. MT(+) carcinomas showed tendency to have highly atypical nuclei, and nuclear staining for Ki-67 antigen was found in a higher percentage of cases than in MT(-) carcinomas. An inverse relationship between MT(+) and ER immunoreactivity was observed. MT expression was not associated with age distribution, menopausal status, tumor size or lymph node metastasis. The overall survival rate in MT(+) cases was worse than in those negative for MT, but no significant association was found. MT(+) was not associated with poor prognosis in total, estrogen receptor-negative or node-negative tumors. These findings suggest that MT expression in breast cancer cells is related to cell-proliferative activity, and that dedifferentiation of carcinoma cells may play a role in induction of MT expression.

Trends of IL-6 and IL-8 levels in patients with recurrent breast cancer: preliminary report.

BackgroundWe reported that IL-6 and IL-8 levels at the beginning of treatment are predictive indicators of response to therapy and prognosis of patients with recurrent breast cancer. The aim of this study was to investigate the trend of IL-6 and IL-8 levels in heavily pretreated patients with recurrent breast cancer.MethodsCytokine level trends in 12 patients heavily pretreated with anthracyclines were studied. Patients were divided into two groups according to the objective response. There were 5 partial response (PR)/no change (NC), and 7 progressive disease (PD) patients. Blood was taken every four weeks. IL-6 was measured by chemiluminescent enzyme immunoassay. IL-8 was measured by ELISA.ResultsThe pretreatment level of IL-6 in the PR/NC group (11.0±2.1 pg/ml) was significantly lower than that (15.3±2.7 pg/ml) in the PD group. However, there was no difference in IL-8 level between the PR/NC group (12.5±5.5 pg/ml) and the PD group (11.5±1.1 pg/ml). IL-6 levels in the PR/NC group were maintained within normal levels or decreased to within normal levels after treatment, while levels of IL-6 in the PD group gradually increased until the time of patient death. A decrease in IL-8 level after treatment was observed in only one patient in the PR/NC group. Mild increase of IL-8 levels was observed in the PD group.ConclusionContinuous elevation of IL-6 levels indicates poor prognosis in heavily pretreated patients with recurrent breast cancer. Combination therapy including agents that reduce IL-6 levels will be a new strategy for aggressively treating recurrent breast cancer.

Mitogen-Activated Protein Kinase Cascade in Breast Cancer

The mitogen-activated protein (MAP) kinase is considered to play a central role in diverse cellular events including carcinogenesis and tumor progression. Indeed, expression of MAP kinase, tyrosine-phosphorylated MAP kinase, and Raf-1 protein was greater in cancerous human tissues than in the surrounding noncancerous glands. In a 7,12-dimethylbenz[a]anthracene-induced rat mammary carcinoma model, estrogen promoted and ovariectomy and antiestrogen, tamoxifen (TAM) inhibited the tumor growth. Ovariectomy suppressed expression of MAP kinase, tyrosine-phosphorylated MAP kinase and Raf-1, whereas estrogen as well as TAM induced expression of MAP kinase and Raf-1 under castrated conditions. Since it was reported that MAP kinase was activated during the progression of breast carcinoma cells, such estrogenic actions of TAM toward the MAP kinase cascade might be responsible for malignant progression.

Serum concentration of hepatocyte growth factor in patients with metastatic breast cancer

The serum concentration of hepatocyte growth factor (HGF) was examined in 34 patients with metastatic breast cancer. Although no significant difference was observed between HGF concentration and the site of metastasis, serum HGF levels were slightly higher in patients with liver metastasis and in patients with multiple metastatic sites than in patients with other lesions. Significantly higher levels of serum HGF were observed in patients with progressive metastasis of breast cancer compared with those with stable metastasis. The patients with high HGF levels exhibited a significantly shorter survival rate than those with low HGF levels. Circulating HGF levels may be a useful indicator for the progression of metastatic lesions and the prognosis of patients with metastatic breast cancer.

Identification of a Novel Chloride Channel Expressed in the Endoplasmic Reticulum, Golgi Apparatus, and Nucleus

MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. UsingMID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.

Activation of Aromatase Expression by Retinoic Acid Receptor-related Orphan Receptor (ROR) α in Breast Cancer Cells IDENTIFICATION OF A NOVEL ROR RESPONSE ELEMENT

Estrogen is a key regulator of the proliferation and differentiation of breast cancer cells. In addition to the estrogen supply from the ovary, estrogen is produced locally from androgen by aromatase. However, the regulation of aromatase gene expression in breast cancer has not yet been fully clarified. Retinoic acid receptor-related orphan receptor (ROR) alpha plays an important role in the differentiation of many organs by regulating the transcription of target genes. Because aromatase and RORalpha are expressed in breast cancer, the effect of RORalpha on aromatase gene expression was studied. RORalpha significantly augmented the expression of aromatase mRNA, particularly those containing exon I.4, in MCF7 cells, and aromatase activities in T47D and MCF7 cells. RORalpha also stimulated the proliferation of these cells. Transient transfection-based reporter gene assays using the promoter at exon I.4 showed that RORalpha augmented the transcription. A series of truncated mutation studies revealed that RORalpha activated the transcription through -147 to +14 bp of the promoter I.4. Furthermore, RORalpha bound to the fragment containing -119 to -107 bp of the promoter in vitro, indicating that this region may contain a novel ROR response element. Chromatin immunoprecipitation assay showed that RORalpha bound to the region containing this site of the promoter I.4 in MCF7 cells. Moreover, we examined clinical samples and found a correlation between RORalpha and aromatase expression. These results suggest that RORalpha directly activates the aromatase expression to accelerate the local production of estrogen, which results in the proliferation of breast cancer cells.Estrogen is a key regulator of the proliferation and differentiation of breast cancer cells. In addition to the estrogen supply from the ovary, estrogen is produced locally from androgen by aromatase. However, the regulation of aromatase gene expression in breast cancer has not yet been fully clarified. Retinoic acid receptor-related orphan receptor (ROR) α plays an important role in the differentiation of many organs by regulating the transcription of target genes. Because aromatase and RORα are expressed in breast cancer, the effect of RORα on aromatase gene expression was studied. RORα significantly augmented the expression of aromatase mRNA, particularly those containing exon I.4, in MCF7 cells, and aromatase activities in T47D and MCF7 cells. RORα also stimulated the proliferation of these cells. Transient transfection-based reporter gene assays using the promoter at exon I.4 showed that RORα augmented the transcription. A series of truncated mutation studies revealed that RORα activated the transcription through −147 to +14 bp of the promoter I.4. Furthermore, RORα bound to the fragment containing −119 to −107 bp of the promoter in vitro, indicating that this region may contain a novel ROR response element. Chromatin immunoprecipitation assay showed that RORα bound to the region containing this site of the promoter I.4 in MCF7 cells. Moreover, we examined clinical samples and found a correlation between RORα and aromatase expression. These results suggest that RORα directly activates the aromatase expression to accelerate the local production of estrogen, which results in the proliferation of breast cancer cells.

Tamoxifen activates CYP3A4 and MDR1 genes through steroid and xenobiotic receptor in breast cancer cells

Cytochrome P450 monooxygenase 3A4 (CYP3A4) and P-glycoprotein, encoded by multidrug resistance 1 (MDR1) gene, are responsible for the metabolism of endogenous steroids, prescribed drugs, and xenobiotics. Both genes are regulated by steroid and xenobiotic receptor (SXR), a member of nuclear hormone receptors. Various endogenous steroids and drugs function as ligands of SXR. Although CYP3A4, MDR1, and SXR are expressed mainly in the liver and the small intestine, these gene products are also expressed in breast cancer cells. Because tamoxifen (TAM) is known to be metabolized by CYP3A4 and P-glycoprotein, weinvestigated the effect of TAM on these SXR-targeted genes in breast cancer cells. Transient transfection-based reporter gene assays showed 4-hydroxy TAM activated the SXR-mediated transcription through CYP3A4 and MDR1 promoters in a ligand- and receptor concentration-dependent manner. We confirmed the binding of 4-hydroxy TAM to SXR by ligand binding assay. Moreover, semiquantitative RT-PCR studies revealed that 4-hydroxy TAM activated the expression of CYP3A4 and MDR1 mRNA in MCF-7 cells. These results suggest that TAM induces CYP3A4 and MDR1 gene expression through SXR, which may affect TAM metabolic pathway in breast cancer cells.

Immunohistochemical Study on the Expression of c-erbB-2 Oncoprotein in Breast Cancer

Expression of the c-erbB-2 oncogene protein was investigated by immunohistochemistry in 110 paraffin-embedded blocks of primary breast cancer. 25 (22.7%) of 110 tumors were stained positively with c-erbB-2 protein antibody. There was no correlation between c-erbB-2 immunostaining and age at diagnosis, menopausal status, hormone receptor status, tumor size, or clinical stage. The tumors with an extensive intraductal component showed a higher incidence of the c-erbB-2 expression than those without. A significantly shorter overall survival was obtained in patients with the expression of c-erbB-2 protein than in patients without. Staining of c-erbB-2 protein is an effective prognostic indicator in patients with breast cancer.

The efficacy of technetium-99m-MIBI scan and intraoperative methylene blue staining for the localization of abnormal parathyroid glands.

The efficacy of the technetium-99m-2-methoxy-isobutylisonitrile (Tc-MIBI) scan and intraoperative methylene blue staining was analyzed in a consecutive series of 15 patients with primary hyperparathyroidism who underwent neck surgical exploration. A total of 17 abnormal parathyroid glands were removed, 7 of which were confirmed histologically as adenomas and 10 as hyperplasias. The Tc-MIBI scan and the thallium-201-technetium-99m subtraction (TI/Tc) scan preoperatively localized 11 (69%) of 16, and 6 (40%) of 15 abnormal parathyroid glands, respectively. The Tc-MIBI scan correctly localized two ectopic abnormal parathyroid glands which were not localized by the Tl/Tc scan or ultrasonography (US). However, it also demonstrated falsepositive accumulations caused by thyroid diseases in two patients. There were 4 abnormal parathyroid glands not detected by the preoperative imaging techniques, whereas all 17 abnormal parathyroid glands were stained with methylene blue, the infusion of which caused no adverse effects or toxicity. In conclusion, Tc-MIBI scanning and intraoperative methylene blue staining are effective techniques for the localization of abnormal parathyroid glands in patients with primary hyperparathyroidism.