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Background: We conducted an open-label, randomized controlled trial evaluating the appropriate treatment duration of leuprorelin acetate 3-month depot, TAP-144-SR (3M), administered postsurgically every 3 months for 2 years versus 3 or more (up to 5) years, in combination with tamoxifen, for 5 years in premenopausal endocrine-responsive breast cancer patients and reported similar survival benefit in the two treatment groups. We hereby present patient-reported quality of life (QOL) data obtained from this trial., Methods: Three self-administered QOL questionnaires (QOL-ACD, QOL-ACD-B, FACT-ES subscale) were used, and the difference in QOL score changes between the two groups was analyzed using a mixed-effects model for repeated measures., Results: Eligible patients (N = 222) were randomly assigned to a 2-year (2YG, N = 112) or 3-or-more-year treatment group (3YG, N = 110). The time courses of the three QOL scores during the trial period were similar in the two groups. The mean changes in the QOL scores from week 96 were largely stable through week 240 in the 3YG, but showed significantly greater improvement in the score changes from week 96 in the 2YG than the 3YG. Symptoms associated with menopause such as hot flashes and sweating contributed to these results. Menstruation recovery was associated with significantly greater improvement of these symptoms in the 2YG than the 3YG., Conclusions: Patient-reported menopause-associated symptoms and QOL improved after discontinuation of the LH-RH agonist administration and menstruation recovery. QOL information should be a consideration in long-term treatment.

Aims: The aim in this study is to evaluate economic value for leuprorelin acetate 6-month depot compared with leuprorelin acetate 3-month depot in Japanese pre-menopausal breast cancer patients from a societal perspective., Methods: The cost analysis was conducted by estimating direct and indirect cost, and intangible costs associated with one 6-month injection compared with two 3-month injections. Claims data were used for the analyses of direct and indirect cost and Medical Fee Schedule Table for direct cost. Discrete choice experiments were conducted by web-based survey to determine the intangible costs. Another web-based survey was also conducted on premenopausal breast cancer patients with injections of leuprorelin acetate, to calibrate the results of discrete choice experiments., Results: The medical costs saved for having one less injection in pre-menopausal breast cancer patients with leuprorelin acetate injection were JPY 6,183. The productivity loss saving was JPY 1,419. An estimation of intangible costs saved for having one less injection of leuprorelin acetate was JPY 58,430, which included the disbenefit due to pain (JPY 8,535), injection site reactions (JPY 44,051), waiting time (JPY 9,595), and subtracting value in medical consultation (JPY 3,751). The total cost saved for having one less injection was JPY 66,032., Limitations: The respondents from the internet panel provided by a survey company do not necessarily reflect a population of Japanese society., Conclusions: Leuprorelin acetate 6-month depot demonstrates a higher value than leuprorelin acetate 3-month depot through saving medical costs and loss of productivity, as well as intangible costs saved for having one less injection when treating pre-menopausal breast cancer patients. In the costs for treating with leuprorelin acetate, the percentage of intangible costs might not be negligible. The intangible costs will probably be actively evaluated to proceed to patient-centered healthcare in society.

Background: Triple-negative breast cancer (TNBC) exhibits biologically aggressive behavior and has a poor prognosis. Novel molecular targeting agents are needed to control TNBC. Recent studies revealed that the non-canonical hedgehog (Hh) signaling pathway plays important roles in the regulation of cancer stem cells (CSCs) in breast cancer. Therefore, the anti-cell growth and anti-CSC effects of the non-canonical Hh inhibitor GANT61 were investigated in TNBC cells., Methods: The effects of GANT61 on cell growth, cell cycle progression, apoptosis, and the proportion of CSCs were investigated in three TNBC cell lines. Four ER-positive breast cancer cell lines were also used for comparisons. The expression levels of effector molecules in the Hh pathway: glioma-associated oncogene (GLI) 1 and GLI2, were measured. The combined effects of GANT61 and paclitaxel on anti-cell growth and anti-CSC activities were also investigated., Results: Basal expression levels of GLI1 and GLI2 were significantly higher in TNBC cells than in ER-positive breast cancer cells. GANT61 dose-dependently decreased cell growth in association with G1-S cell cycle retardation and increased apoptosis. GANT61 significantly decreased the CSC proportion in all TNBC cell lines. Paclitaxel decreased cell growth, but not the CSC proportion. Combined treatments of GANT61 and paclitaxel more than additively enhanced anti-cell growth and/or anti-CSC activities., Conclusions: The non-canonical Hh inhibitor GANT61 decreased not only cell growth, but also the CSC population in TNBC cells. GANT61 enhanced the anti-cell growth activity of paclitaxel in these cells. These results suggest for the first time that GANT61 has potential as a therapeutic agent in the treatment of patients with TNBC.

Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between aromatase expression and aromatase inhibitors are warranted.

Estradiol (E2) increases not only the cell growth but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. It has been suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of CSC proportion in ER-positive breast cancer cells. We studied anti-CSC activity of a non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. Effects of GANT61 on the cell growth, cell cycle progression, apoptosis and CSC proportion were investigated in four ER-positive breast cancer cell lines. CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of GANT61 with antiestrogens on the anti-cell growth and anti-CSC activities were investigated. E2 significantly increased the cell growth and CSC proportion in all ER-positive cell lines. E2 increased the expression levels of glioma-associated oncogene (GLI) 1 and/or GLI2. GANT61 decreased the cell growth in association with a G1-S cell cycle retardation and increased apoptosis. GANT61 decreased the E2-induced CSC proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2-induced cell growth and CSC proportion. Combined treatments of GANT61 with antiestrogens additively enhanced anti-cell growth and/or anti-CSC activities in some ER-positive cell lines. In conclusion, the non-canonical Hh inhibitor GANT61 inhibited not only the cell growth but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced anti-cell growth and/or anti-CSC activities of antiestrogens in ER-positive cell lines., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Leuprorelin acetate, a luteinizing hormone-releasing hormone agonist, is used worldwide in premenopausal women with hormone receptor-positive breast cancer. This study was conducted to assess the non-inferiority of the 6-month depot formulation, TAP-144-SR (6M) 22.5 mg to the 3-month depot formulation, TAP-144-SR (3M) 11.25 mg in postoperative, premenopausal patients with hormone receptor-positive breast cancer., Methods: This was a 96-week phase III, randomized, open-label, parallel-group comparative study. All patients concomitantly received oral tamoxifen (20 mg daily). The primary endpoint was the suppression rate of serum estradiol (E 2 ) to the menopausal level (≤30 pg/mL) from Week 4 through Week 48., Results: In total, 167 patients were randomized to receive TAP-144-SR (6M) (n = 83) or TAP-144-SR (3M) (n = 84) and the E 2 suppression rate was 97.6 and 96.4 %, respectively. The estimated between-group difference was 1.2 % (95 % confidence interval -5.2 to 7.8). The non-inferiority of TAP-144-SR (6M) to TAP-144-SR (3M) for E 2 suppression was confirmed. As for safety, common adverse events were hot flush and injection site reactions including induration, pain, and erythema in both treatment groups, which were of ≤Grade 2 in severity and not serious. No significant between-group differences in safety profiles and tolerability were observed., Conclusions: TAP-144-SR (6M) was not inferior to TAP-144-SR (3M) for its suppressive effect on serum E 2 . TAP-144-SR (6M) was also as well tolerated as TAP-144-SR (3M)., Competing Interests: J. Kurebayashi received advisory/consultation fees from Takeda Pharmaceutical Company Limited and research funding from Takeda Pharmaceutical Company Limited, Eisai Company Limited, Chugai Pharmaceutical Company Limited and AstraZeneca K.K. T. Toyama received a research funding from Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Healthcare Co., Ltd. and Novartis Pharma K.K. S. Sumino, E. Miyajima and T. Fujimoto are employees of Takeda Pharmaceutical Company Limited. This work was supported by Takeda Pharmaceutical Company Limited.

The membrane-anchored glycoprotein RECK negatively regulates multiple metalloproteinases and is frequently downregulated in tumors. Forced RECK expression in cancer cells results in suppression of tumor angiogenesis, invasion, and metastasis in xenograft models. A previous methylome study on breast cancer tissues detected inverse correlation between RECK CpG methylation (in an intron-1 region) and relapse-free survival. In this study, we focused on another region of the RECK CpG island (a promoter/exon-1 region) and found an inverse correlation between its methylation and RECK-inducibility by an HDAC inhibitor, MS275, among a panel of breast cancer cell lines (n=15). In clinical samples (n=62), RECK intron-1 methylation was prevalent among luminal breast cancers as reported previously (26 of 38 cases; 68%) and particularly enriched in tumors of the ER+PR- subclass (10 of 10 cases) and of higher histological grades (Grade 2 and 3; 28 of 43 cases; P=0.006). In about a half of these cases, promoter/exon-1 methylation was absent, and hence, RECK may be inducible by certain drugs such as MS275. Our results indicate the value of combined use of two RECK methylation markers for predicting prognosis and drug-sensitivity of breast cancers.

Background: Luteinizing hormone-releasing hormone (LH-RH) agonists provide effective adjuvant treatment for premenopausal women with endocrine-responsive breast cancer. Here, we investigated appropriate treatment durations of an LH-RH agonist, leuprorelin., Methods: We conducted an open-label, randomized controlled pilot study to evaluate the safety and efficacy of leuprorelin subcutaneously administered every-3-months for 2 versus 3 or more, up to 5 years, together with daily tamoxifen for 5 years in premenopausal endocrine-responsive breast cancer patients. Primary endpoints were disease-free survival (DFS) and safety., Results: Eligible patients (N = 222) were randomly assigned to receive leuprorelin for either 2 years (N = 112) or 3 or more years (N = 110) with tamoxifen for 5 years after surgery. Leuprorelin treatment for 3 or more years provided no significant difference in DFS rate over 2 years: 94.1 versus 91.8 % at 144 weeks (3 years) after the second year (week 96) and 90.8 versus 90.4 % at the fifth year (week 240). The overall survival rate was 100 % for both groups during the third through fifth year study period. There were no significant differences in the incidence of adverse events (AEs) between the 2 groups: most AEs were rated grade 1 or 2., Conclusions: Adjuvant leuprorelin treatment for 3 or more years with tamoxifen showed a survival benefit and safety profile similar to that for 2 years in premenopausal endocrine-responsive breast cancer patients. No new safety signal was identified for long-term leuprorelin treatment. Longer follow-up observation is needed to determine the optimal duration of leuprorelin treatment.

Background: Eribulin mesylate (eribulin), a non-taxane microtubule dynamic inhibitor, has been widely used in the treatment of patients with advanced or metastatic breast cancer. The combined antitumor and anticancer stem cell (CSC) activities of eribulin with endocrine therapeutic agents have not yet been examined in breast cancer cells. We herein investigated the combined effects of eribulin and antiestrogens., Methods: A panel of eight breast cancer cell lines, including five estrogen receptor (ER)-positive and three ER-negative cell lines, was used. These cells were treated with eribulin and/or the antiestrogen, 4-hydroxytamoxifen or fulvestrant. Their growth inhibitory activities and effects on cell cycle progression, apoptosis, and the CSC population were investigated. CSCs were detected using the CD44/CD24/EpCAM, Aldefluor, and mammosphere assays., Results: The 50% growth inhibitory concentrations of eribulin were 0.38-2.64 nM for the eight cell lines tested. Eribulin exhibited significant antitumor activity under estrogen-supplemented conditions in ER-positive breast cancer cells. The combined antitumor activity of eribulin with an antiestrogen was evaluated using the combination index. The combination index was 0.43-1.46 for ER-positive cell lines. The additive antitumor effect of eribulin with 4-OHT was only significant in MCF-7 cells. Eribulin induced the accumulation of G2/M and apoptosis, while antiestrogens induced the retardation of G1-S cell cycle and apoptosis, respectively. Estrogen markedly increased the proportion of CSCs, whereas antiestrogens inhibited increases in ER-positive cell lines. Moreover, eribulin decreased the proportion of CSCs in either ER-positive or ER-negative cell lines. The combined treatment of eribulin with an antiestrogen did not additively decrease the proportion of CSCs in ER-positive cell lines., Discussion: The results of the present study demonstrated that eribulin had potent antitumor effects on estrogen-stimulated ER-positive breast cancer cells and the combined treatment of eribulin with an antiestrogen resulted in a weakly additive antitumor effect. We herein suggested for the first time that eribulin exhibited anti-CSC effects on either ER-positive or ER-negative breast cancer cells.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

Background: Adjuvant trastuzumab has been routinely used in HER2-positive operable breast cancer patients. Prognostic factors remain to be well characterized in these patients and might correlate with primary and/or acquired resistance to trastuzumab., Patients and Methods: The study subjects were 78 HER2-positive operable breast cancer patients treated with adjuvant chemotherapy followed by 1-year trastuzumab between 2005 and 2010 in our institute. All breast tumors showed a HercepTest score of 3+ or that of 2+ and positive fluorescence in situ hybridization. Expression levels of HER1, phosphorylated HER2 (pY1248), HER3, HER4, and p53 were assessed by immunohistochemistry. Prognostic factors were investigated with univariate and multivariate analyses using the Kaplan-Meier/log-rank test and Cox proportional hazards model, respectively., Results: The median age and follow-up period of the patients were 54 years and 39 months, respectively. The mean tumor size was 2.1 cm and the node-positive rate was 42 %. Eight patients had recurrent diseases but no patient died of cancer. Univariate analysis revealed that pHER2 positivity was only a significantly worse prognostic factor for relapse-free survival (RFS) (P = 0.049). A HercepTest score of 2+ and high expression level of p53 showed a trend. Multivariate analysis revealed three biological markers: pHER2 positivity [hazard ratio (HR) = 11.6, 95 % confidence interval (CI) 1.3-111.1, P = 0.031], p53 positivity (HR = 6.4, 95 % CI 1.0-40.0, P = 0.047) and a HercepTest score of 2+ (HR = 8.6, 95 % CI 1.6-45.2, P = 0.011) to be worse prognostic factors for RFS. Notably, three out of five patients with breast tumors expressing HER2 at a score of 2+ and pHER2 had recurrent diseases. Interestingly, the expression level of pHER2 significantly correlated with the expression levels of HER2 and HER3 in HER2-positive breast tumors., Conclusions: This retrospective cohort study suggests that a lower expression level of HER2 and high expression levels of pHER2 and p53 may indicate a worse prognosis in HER2-positive breast cancer patients treated with trastuzumab and chemotherapy. Further studies are needed to evaluate pHER2 expression in HER2-positive breast cancer as a prognostic and/or predictive marker.

Purposes: We conducted a study to analyze the clinicopathological characteristics of breast cancer in Japan registered to the Japanese Breast Cancer Registry of the Japanese Breast Cancer Society (JBCS). Trends in the management of breast cancer patients in Japan were also analyzed., Patients and Methods: More than 250,000 breast cancer patients were registered to the JBCS registry between 2004 and 2011. Demographic and clinicopathological factors in newly diagnosed primary breast cancer patients were registered to the JBCS through the Web-based system from affiliated institutes nationwide., Results: Two distinct peaks were observed, in patients in their late 40s and early 60s, in the population-adjusted age distribution of breast cancer patients. An increased rate of screen-detected breast cancer may contribute to an earlier detection of breast cancer and increased rate of non-invasive ductal carcinoma. The positive rate of either ER or PgR appears to have increased in recent years. The annual rates of patients treated with breast-conserving surgery increased until 2006, but these increases stopped in 2007 and thereafter plateaued at approximately 60 %. The annual rates of patients treated with sentinel lymph node dissection alone have steadily increased. The annual rates of patients treated with preoperative trastuzumab plus chemotherapy have also increased, as well as those treated with postoperative aromatase inhibitors. The annual rates of patients treated with postoperative anthracycline-containing regimens have decreased, whereas those treated with postoperative taxane-containing regimens have increased. The postoperative use of trastuzumab has markedly increased since 2007., Conclusion: Although this study was based on the registry database, several unique clinicopathological characteristics of breast cancer in Japan have been unveiled. Our results suggest that recent trends in the management of breast cancer patients in Japan were strongly followed by clinical evidence that originated from a number of clinical trials worldwide.

The Cell Cycle Profiling - Risk Score (C2P-RS) based on CDK1 and CDK2 specific activities was significantly associated with relapse in breast cancers. We evaluated the prognostic value of the C2P-RS classification using a Japanese cohort including node-negative, hormone receptor-positive breast cancers treated with adjuvant endocrine therapy alone as systemic therapy. Of 266 patients, 22 (8.3%) relapsed within 5 years after surgery. The distribution of each C2P-RS group was 71.8% in the low group, 12.0% in the intermediate group, and 16.2% in the high group. The 5-year relapse-free survival rate in the low C2P-RS group (97.3%) was significantly higher than that in the intermediate C2P-RS group (84.3%) or the high C2P-RS group (74.4%) (P < 0.001). The univariate analysis demonstrated that age, tumor size, histologic grade, and HER2 had no significant correlations with relapse but the C2P-RS classification (P < 0.001) and Ki-67 (P = 0.009) were significantly associated with relapse. Multivariate analysis showed only that the C2P-RS classification was a significant independent prognostic indicator. The C2P-RS classification might be a significant predictor of earlier recurrence in node-negative, hormone receptor-positive breast cancers treated with endocrine therapy., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Whether postoperative chemotherapy should be added to endocrine therapy or not is an important issue in patients with hormone receptor-positive and human epidermal growth factor receptor (HER)2-negative breast cancer. To identify patients who should be treated with additional chemotherapy, prognostic factors were investigated in breast cancer patients postoperatively treated with endocrine therapy alone., Patients and Methods: Tumor samples and clinicopathological data were collected from patients who underwent curative surgery and were postoperatively treated with endocrine therapy alone between 1999 and 2003 in three different institutes. Expression levels of estrogen receptor (ER), progesterone receptor (PgR), and HER2 in primary tumors were centrally retested. Patients with ER-negative and/or HER2-positive tumors and/or with unknown nodal status were excluded from the study subjects. Immunohistochemical analysis of Ki67, HER1, insulin-like growth factor-1 receptor, and aldehyde dehydrogenase-1 was also performed. Prognostic factors were investigated by univariate and multivariate analyses., Results: A total of 261 patients were the subjects of this study. The median age was 59 years old, the mean tumor size was 1.9 cm, the node-positive rate was 20 %, and 65 % received tamoxifen alone. Distant metastases were observed in 11 patients at a median follow-up of 98 months, and four patients had died of breast cancer at a median follow-up of 99 months. Univariate analysis showed that marked lymphovascular invasion (LVI), PgR negativity, high Ki67 labeling index (LI), and high nuclear grade were significantly worse prognostic factors for distant metastasis. Multivariate analysis revealed that marked LVI [hazard ratio (HR) 21.8] and PgR negativity (HR 10.3) were independently worse prognostic factors for distant metastasis, respectively. Multivariate analysis also revealed that marked LVI (HR 287.3), PgR negativity (HR 25.1), and high Ki67 LI (HR 19.6) were independently worse prognostic factors for breast cancer-specific death, respectively., Conclusions: The results of this multi-institute cohort study indicated that endocrine therapy alone could not prevent distant metastasis in breast cancer patients with PgR-negative tumors and/or with tumors showing marked LVI or high cell proliferation. These patients may need postoperative adjuvant chemotherapy in addition to endocrine therapy.

Background: In Japan, there are still no reports of long-term outcome for hypofractionated radiotherapy to the whole breast after breast-conserving surgery (BCS). We report our institution's results from evaluation of the efficacy and safety of hypofractionated radiotherapy for Japanese women., Methods: Data in the medical records of 327 patients were retrospectively reviewed. The patients were treated with hypofractionated radiotherapy between January 2003 and December 2006 at the Kawasaki Medical School Hospital and were followed for more than 3 years. The median age was 54 years old (the age range was 28-80 years). The whole breast was irradiated with a total dose of 42.56 Gy/16 fx with boost irradiation to positive margins. Adjuvant therapy consisted of chemotherapy and/or hormone therapy and was administered to 300 patients, based on their stage or pathological findings., Results: Follow-up periods ranged from 21 to 92 months; the median follow-up period was 60 months. At 5-year follow-up, overall survival, cause-specific survival, relapse-free survival, and local control were 96.0, 97.5, 95.3, and 99.7% respectively. Grade 2 radiation pneumonitis occurred in five patients. Grade 2 radiation dermatitis occurred in 17 patients. Severe late complications were not observed., Conclusions: In our study, hypofractionated radiotherapy led to good results without severe toxicity. We believe hypofractionated radiotherapy after BCS is safe and efficient treatment for Japanese women.

The patient was a 44-year-old woman who exhibited a diffuse goiter during health screening. Her medical history did not include any significant medication-based treatment. An echographic examination detected a solid cystic tumor, which measured 21 × 14 × 10 mm, in her right thyroid lobe; however, she displayed normal thyroid function. After fine-needle aspiration cytology had been performed with a 22 G injection needle, the patient immediately complained of compression and pain extending from the front of her neck to her lower chin, which was not accompanied by dyspnea. A second echographic examination revealed diffuse and edematous enlargement and increased internal blood flow in the bilateral thyroid lobes as well as a thyroid nodule. We immediately iced the patient's neck and administered 125 mg methylprednisolone via an intravenous infusion. Within one hour, her symptoms had markedly improved, but acute pain remained. Thus, we continued the steroid (prednisone) treatment, but the dose was gradually reduced from 10 mg/day to 5 mg/day at 1 week after the patient's symptoms disappeared. The mechanism responsible for the patient's condition remains unclear.

Background: Although the poly adenosine diphosphate (ADP)-ribose polymerase (PARP) inhibitor olaparib is known to have potent antitumor activity in BRCA-related breast cancer cells, a limited number of preclinical and clinical studies have shown antitumor activity of olaparib in non-BRCA-related breast cancer. We investigated antitumor activity of olaparib in breast cancer cell lines derived from patients with nonfamilial sporadic breast cancer., Methods: Effects of olaparib alone or in combination with five different chemotherapeutic agents on cell growth, cell cycle progression, apoptosis, and proportion of cancer stem cells using the mammosphere assay and CD44/CD24/ESA cell surface marker assay were investigated in a panel of six sporadic breast cancer cell lines. Extracellular-signal-regulated kinase (ERK) phosphorylation was also investigated to elucidate action mechanisms of olaparib., Results: Olaparib inhibited the growth of two estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell lines and two ER-negative and HER2-negative breast cancer cell lines (50% growth inhibitory concentrations 1.3-3.0 μM) associated with G2/M accumulation and induction of apoptosis. In contrast, two HER2-positive cell lines were resistant to olaparib. Interestingly, olaparib significantly decreased the proportion of putative cancer stem cells in either sensitive or resistant cell lines. In addition, olaparib increased expression of p-ERK. Combined treatments of olaparib with a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 completely suppressed expression of p-ERK. These treatments also inhibited the G2/M accumulation and apoptosis induction by olaparib. Among five chemotherapeutic agents commonly used for breast cancer treatment, only an irinotecan metabolite SN38 showed additive antitumor activity with olaparib. Importantly, the combined treatment enhanced the increase in G2/M accumulation and apoptosis induction as well as a decrease in the proportion of cancer stem cells., Conclusions: This study has indicated for the first time that the PARP inhibitor olaparib has substantial antitumor and anticancer stem cell activity in breast cancer cell lines of nonfamilial origin. Upregulation of p-ERK might explain, at least in part, antitumor and anticancer stem cell activity of olaparib. Combined treatment of olaparib with irinotecan might be effective in treatment of non-BRCA-related breast cancer.

Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7) was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P = 0.04, Fisher's exact test). Thus, we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

It is speculated that estrogens play important roles in the male breast carcinoma (MBC) as well as the female breast carcinoma (FBC). However, estrogen concentrations or molecular features of estrogen actions have not been reported in MBC, and biological significance of estrogens remains largely unclear in MBC. Therefore, we examined intratumoral estrogen concentrations, estrogen receptor (ER) α/ERβ status, and expression profiles of estrogen-induced genes in MBC tissues, and compared these with FBC. 17β-Estradiol concentration in MBC (n = 4) was significantly (14-fold) higher than that in non-neoplastic male breast (n = 3) and tended to be higher than that in FBC (n = 7). Results of microarray analysis clearly demonstrated that expression profiles of the two gene lists, which were previously reported as estrogen-induced genes in MCF-7 breast carcinoma cell line, were markedly different between MBC and FBC. In the immunohistochemistry, MBC tissues were frequently positive for aromatase (63 %) and 17β-hydroxysteroid dehydrogenase type 1 (67 %), but not for steroid sulfatase (6.7 %). A great majority (77 %) of MBC showed positive for both ERα and ERβ, and its frequency was significantly higher than FBC cases. These results suggest that estradiol is locally produced in MBC tissue by aromatase. Different expression profiles of the estrogen-induced genes may associate with different estrogen functions in MBC from FBC, which may be partly due to their ERα/ERβ status.

Background: It has been confirmed by several clinical trials that the fentanyl patch causes less adverse events than sustained-release oral morphine, and after rotation. However, there has been no evidence comparing the fentanyl patch with controlled-release oral oxycodone in terms of adverse events., Purpose: We prospectively investigated the reduced effects of adverse events caused by sustained-release oral morphine and controlled-release oxycodone after rotating to the fentanyl patch in patients with metastatic breast cancer., Method: Metastatic breast cancer patients requiring sustained-release oral morphine or controlled-release oral oxycodone(n=9, 2 taking oral morphine, 7 taking oral oxycodone, mean age, 57. 5 years)were recruited. Those experiencing adverse events from oral morphine or oral oxycodone were administered a fentanyl patch., Results: The pain score was reduced significantly at the 4th week. The fentanyl patch was associated with significantly less nausea, vomiting, constipation, sleepiness and dizziness over the study period., Conclusion: This study suggested that the fentanyl patch can reduce adverse events caused by sustained-release oral morphine as well as controlled-release oral oxycodone.

Background: More than 80% of all thyroid cancers, the most common endocrine malignancy, are papillary thyroid cancer (PTC). It is well established that CITED1 (Cbp/p300 Interacting Transactivators with glutamic acid [E] and aspartic acid [D]-rich C-terminal domain) mRNA is characteristically overexpressed in PTC. Our previous study suggested a positive association of BRAF mutation with CITED1 overexpression. However, the mechanism of CITED1 expression in PTC remains to be elucidated. In the present study, we analyzed whether aberrant methylation of CITED1 gene promotes CITED1 overexpression in PTC., Method: CITED1 mRNA expression levels were analyzed by quantitative polymerase chain reaction in three PTC-derived cell lines, TPC1, K1, and KTC-1, and in surgically dissected PTC and surrounding normal tissues from 19 patients. The BRAF mutation status of the cells and clinical specimens was determined by direct sequencing. The methylation status of the deoxycitidine-phosphate-deoxyguanosine dinucleotides (CpGs) in the CITED1 promoter was analyzed by the bisulfite-sequencing method using genomic DNA. Finally, the expression of CITED1 mRNA in TPC1 cells, when subjected to pharmacological inhibition of methylation, was analyzed., Results: CITED1 mRNA was expressed at lower levels in TPC1 than in K1 and KTC-1 cells. A BRAF mutation was present in K1 and KTC-1 cells, but not in TPC1 cells. CITED1 promoter was hypomethylated in K-1 and KTC-1 cells, but not in TPC1 cells. In surgically dissected specimens, the mean expression level of CITED1 mRNA was 30-fold higher in PTC tissues than in normal tissues. CpGs in the CITED1 promoter were more heavily methylated in normal tissues than in PTC tissues. In PTC specimens without a BRAF mutation, two CpGs were more heavily methylated than in PTC specimens with the BRAF V600E mutation. Pharmacological inhibition of methylation in TPC1 cells by 5'-aza-2'-deoxycitidine resulted in increased expression of CITED1 mRNA., Conclusion: Hypomethylation of the CpGs in the promoter region of CITED1 is associated with higher expression of CITED1 mRNA in PTC tissues, consistent with the hypothesis that epigenetic regulation is involved in the overexpression of CITED1. This hypothesis is supported by pharmacologic inhibition studies in TPC1 cells.

Background. We retrospectively analyzed whether poor differentiation is the independent prognostic factor for thyroid carcinoma or not. Methods. The subjects were 29 patients with PDTC who were treated between April 1996 and March 2006 to compare with those of well-differentiated papillary carcinoma patients (n = 227). Results. The relapse free (RFS), distant relapse-free survival and cause-specific survival, rates were significantly lower in patients with PDTC (P < .0001, P < .001, and P < .05). After classification into focal (<10%) and diffuse type (over 10%) of PDTC, there were no significant differences in RFS and cause-specific survival due to component type or proportion of poorly differentiated component. On multivariate analysis, poor differentiation (P < .0005, RR = 4.456, 95% CI; 1.953-10.167) and extrathyroidal infiltration (P < .05, RR = 2.898, 95% CI; 1.278-6.572) showed a significant impact on DFS, and poor differentiation (P < .05, RR = 9.343, 1.314-66.453) and age (P < .005, RR = 1.306, 1.103-1.547) significantly impacted cause-specific survival. Conclusion. Poor differentiation was an independent factor for survival. Distant relapse was significantly more common among PDTC patients, and systemic therapy might be warranted.

A 53-year-old woman with left breast tumor was diagnosed as bilateral breast cancer(left; T3N3M0, Stage III C/right; T2N0M0, Stage II )in our hospital, both of which were revealed as invasive ductal carcinoma shown to be ER-negative, PgR negative and HER2-positive by core needle biopsy. In December 2004, paclitaxel and trastuzumab combination therapy was tried, but she went into shock just during administration of paclitaxel, and this therapy was discontinued. After that the triweekly CTF therapy was tried as an anthracycline containing regimen, and the lymph node metastases obtained a complete response after a month and a 38. 5% reduction of left primary breast tumor, which was the best response observed after three months. Time to progression was prolonged to 7 months(9 cycles). Although febrile neutropenia occurred in the first cycle, the therapy could be continued safely thereafter as an outpatient. Anthracycline-containing regimens are likely to be avoided because of the difficulty of combining with trastuzumab in the treatment of HER2 overexpressing advanced/metastatic breast cancer. But the CTF therapy of less cardiotoxicity and less alopecia, can expect longer use and better QOL as an alternative for HER2 overexpressing advanced/metastatic breast cancer patients.

Background: Recent studies have suggested that the Src inhibitor dasatinib preferentially inhibits the growth of breast cancer cells of the basal-like subtype. To clarify this finding and further investigate combined antitumor effects of dasatinib with cytotoxic agents, a panel of breast cancer cell lines of various subtypes was treated with dasatinib and/or chemotherapeutic agents., Methods: Seven human breast cancer cell lines were treated with dasatinib and/or seven chemotherapeutic agents. Effects of the treatments on c-Src activation, cell growth, cell cycle, apoptosis and the proportion of aldehyde dehydrogenase (ALDH) 1-positive cells were examined., Results: The 50%-growth inhibitory concentrations (IC50s) of dasatinib were much lower in two basal B cell lines than those in the other cell lines. The IC50s of chemotherapeutic agents were not substantially different among the cell lines. Dasatinib enhanced antitumor activity of etoposide in the basal B cell lines. Dasatinib induced a G1-S blockade with a slight apoptosis, and a combined treatment of dasatinib with etoposide also induced a G1-S blockade in the basal B cell lines. Dasatinib decreased the expression levels of phosphorylated Src in all cell lines. Interestingly, dasatinib significantly decreased the proportion of ALDH1-positive cells in the basal B cell lines but not in the other cell lines., Conclusions: The present study indicates that dasatinib preferentially inhibits the growth of breast cancer cells of the basal B subtype associated with a significant loss of putative cancer stem cell population. A combined use of dasatinib with etoposide additively inhibits their growth. Further studies targeting breast cancers of the basal B subtype using dasatinib with cytotoxic agents are warranted.

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.

Since the concept of gene profile-based intrinsic subtypes was proposed, various studies on pathological characteristics have been performed. Particularly, triplenegative (TN) breast cancer, which is negative for all hormone receptors [estrogen receptor (ER) and/or progesterone receptor (PgR) and human epidermal growth factor 2 (HER2)], has been attracting attention because effects of endocrine and targeting therapies cannot be anticipated and thus selecting a treatment method is difficult. TN cancer accounts for about 10%-15% of all invasive breast cancer cases in Japanese, which is significantly lower than the incidence reported in the United States. Cytokeratin (CK) 5/6 or epidermal growth factor receptor (EGFR) is positive in 80%, being classified as basal-like carcinoma, but it should be understood that TN breast cancer and basal-like carcinoma are not necessarily the same. Criteria for positivity judgment of ER, PgR, and HER2 were established to select treatment in cases positive for each marker, and greater importance is attached to strict accuracy control. Inversely, the level of negative findings to judge TN varies among the judgment criteria. In any case, the prognosis of TN breast cancer is poor. Pathologically, TN breast cancer shows certain morphological characteristics, such as high grade and a pushing margin, and abnormalities of BRCA1 and p53 are frequently noted. At present, as no effective therapeutic strategy has been established for TN breast cancer, further clarification of the molecular biological characteristics of such cancers is needed. In addition, the incidence of TN-type ductal carcinoma in situ (DCIS) is low, suggesting that TN does not remain preinvasive DCIS for a prolonged period and that it transforms to invasive cancer in an early stage. Because mammary gland basal cells have characters of progenitor or stem cells that differentiate into both luminal epithelium and myoepithelial cells, these cells may be utilized for the differential diagnosis of the benignity or malignancy of intraductal lesions in routine pathological practice. As proliferation markers, such as Ki-67, and multiple gene arrays for gene signature are also utilized to select adjuvant therapy, analysis may progress further in the future.

Background: Triple negative (TN) breast cancer is defined as a subtype that is negative for estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). To clarify the characteristics of TN breast cancer, surveillance data of the Registration Committee of the Japanese Breast Cancer Society were analyzed., Method: Of 14,748 cases registered in 2004, 11,705 (79.4%) were examined for ER, PgR, and HER2. Of these, the most prevalent (53.8%) was a hormone-responsive subtype with ER positive/PgR positive/HER2 negative, followed by TN subtype (15.5%)., Results: The proportion of postmenopausal patients was relatively high in the TN subtype. This cancer was diagnosed at a slightly advanced stage and with more cases positive for lymph node metastases than other subtypes. Morphologically, the TN subtype was more frequently classified as solid-tubular carcinoma. Mucinous, tubular, or secretary carcinomas were frequently found in the hormone receptor positive/HER2 negative subtype, while squamous cell carcinoma, spindle cell carcinoma, and metaplastic carcinoma with bone/cartilage metaplasia were very frequently found in the TN group. Apocrine carcinoma was also found very frequently in the TN group. Selection of chemotherapy was not based on receptor subtypes, but was determined by the degree of tumor progression., Conclusions: Although TN types are similar to basal-like breast tumor, as determined by gene profiling, their diagnosis needs verification by determination of the level of epidermal growth factor receptor or cytokeratin 5/6 expression. TN type should be examined further for immunohistochemical features and analyzed for prognostic details in this cohort.

Predictive factors for anthracycline-based chemotherapy have yet to be incorporated into daily practice. Meta-analyses of studies using anthracycline-based treatment regimens have shown an improved prognosis for human epidermal growth factor receptor type 2 (HER2)-positive tumors, but not for HER2-negative tumors compared with results of non-anthracycline regimens. Currently it is believed that the positive association between HER2 status and anthracycline sensitivity is indirect, that is, their association may be mediated through topoisomerase II alpha (TOP2A), a target molecule of anthracyclines, since TOP2A is near HER-2 and co-amplification of the TOP2A gene frequently occurs in HER2-amplified tumors. This strongly suggests that TOP2A gene amplification is a predictive factor for anthracyline-based regimens. The Collaborative Study Group of Scientific Research of the Japanese Breast Cancer Society has demonstrated that TOP2A-positive and BRCA1-negative subsets evaluated by immunohistochemical staining show a significantly higher pathological complete response when treated with preoperative epirubicin-containing regimens. Combining these findings with the observation that triple-negative tumors and basal-like tumors respond to anthracycline treatment suggests that not only HER2-positive tumors but also a distinct subset of HER2-negative tumors may be sensitive to anthracycline-based regimens.

Background: Preoperative lymphoscintigraphy is commonly used in sentinel lymph node biopsy (SLNB) for patients with early breast cancer; however, its significance to predict SLN metastasis remains to be determined., Patients and Methods: Sixty patients were enrolled in a feasibility study of SLNB. Patients with clinically node-negative breast cancer were eligible for this study. Dynamic lymphoscintigraphy was performed before SLNB. All patients underwent SLNB followed by axillary lymph node dissection., Results: A dual mapping procedure using isotope and dye injections was performed. SLNs were identified in 59 of 60 patients (98.3%), with a node-positive rate of 41.7% and a false-negative rate of 1.7%. No SLN was identified in 4 of 60 patients (6.7%) on preoperative lymphoscintigraphy. Interestingly, abnormal accumulation of the radiotracer close to hot spots was observed in 29 of 56 patients (51.8%). Lymph node metastases were detected in 18 of 29 patients (62.0%) with this pattern and 5 of 27 patients (18.5%) without this pattern (P < 0.05). Micrometastases were more frequently detected in node-positive patients without this pattern than in those with this pattern (80 vs. 16.7%). Diagnostic parameters of this pattern to predict SLN metastases, including micrometastases, were 62.1% for sensitivity, 81.5% for specificity, and 71.4% for accuracy., Conclusions: Abnormal accumulation of the radiotracer close to radioactive spots may indicate SLN metastasis. When dynamic lymphoscintigraphy shows this pattern, surgeons should consider the presence of SLN metastasis and carefully remove additional lymph nodes surrounding radioactive lymph nodes so as not to leave metastatic SLNs behind.

Purpose: Our previous study indicated that concurrent administration of 4-OH-tamoxifen (TAM) and 5-fluorouracil (5-FU), but not doxorubicin (Dox), resulted in additive antitumor effects on endocrine-responsive breast cancer cells. We further clarified the effects of combined administration of endocrine therapy with chemotherapeutic agents in this study., Methods: Concurrent treatment with 4-OH-TAM and paclitaxel (Ptx) was investigated in estrogen receptor (ER)-positive breast cancer cells. Additionally, the combined effects of estrogen depletion from culture medium mimicking estrogen ablative therapy with 5-FU, Dox, and Ptx were investigated., Results: Concurrent treatment with 4-OH-TAM and Ptx yielded less than additive antitumor effects in ER-positive breast cancer cells, as observed with Dox in our previous study. More interestingly, estrogen depletion with 5-FU, but with neither Dox nor Ptx, yielded additive antitumor effects on these cells. We also performed preliminary experiments to elucidate the mechanisms of action responsible for the combined antitumor effects observed. Ptx upregulated the level of expression of one of the molecules related to TAM resistance, Eph-A2, as observed with Dox in our previous study. Estrogen depletion down-regulated the level of expression of one of the molecules related to 5-FU resistance, thymidylate synthase, as observed with 4-OH-TAM in our previous study., Conclusions: These findings, together with those of our previous study, suggest that concurrent treatment with endocrine therapy, administration of TAM, or estrogen ablative therapy and 5-FU but neither Dox nor Ptx may yield additive antitumor effects on endocrine-responsive breast cancer.

Background: Though irinotecan hydrochloride(CPT-11)was approved in Japan in 1994, there have been few reports since that evaluated the efficacy of CPT-11. The position of this agent in the treatment of patients with metastatic breast cancer(MBC)is not definite. In addition, no report has been published to date about CPT-11 and trastuzumab combination therapy., Purpose: To evaluate retrospectively the efficacy of CPT-11 and trastuzumab combination therapy as salvage treatment in patients with human epidermal growth factor receptor 2 (HER2)overexpressing MBC., Patients and Method: We examined ten cases who received this therapy against MBC since February 2002 till March 2007 in our hospital. Overall response rate, change of tumor markers, time to treatment failure (TTF), time to progression( TTP), overall survival (OS)after start of CPT-11 and adverse events were examined for efficacy and tolerability., Results: Median age was 57 (40-67)and median number of prior chemotherapies was 5(2-9). Though the overall response rate was 20%, some tumor reduction was observed totally in seven cases. CEA and CA15-3 were decreased in 78%(7/9)and 63%(5/8) of cases, respectively. Median TTF, TTP and OS were 3, 4, and 6 months, respectively. Adverse events included three cases of severe neutropenia, one of whom died of treatment-related sepsis. Slight diarrhea occurred in seven and severe nausea occurred in two cases. Dose modification of CPT-11 was necessary in 5 cases, and three discontinued CPT-11 administration, mainly due to easy fatigability, nausea and neutropenia. During the therapy, four cases were all inpatients, and 6 eventually became outpatients., Discussion: This therapy for patients with HER2 overexpressing metastatic cancer resistant to multi-agents demonstrated a good result in terms of antitumor effect. But high tolerability could not be documented by our experience with this therapy. It was supposed that the risk management with prediction of adverse events and preparation of better supportive therapy could make for the higher tolerability of this therapy for more effective clinical use.

Taxanes are approved for the treatment of breast cancer that has spread to the lymph nodes, following surgery and doxorubicin containing chemotherapy. Taxanes have improved the survival of breast cancer patients, especially in estrogen receptor (ER) negative population in clinical settings. This time we examined the relationship between chemosensitivity to Taxanes and expresson of ERalpha in breast cancer cell lines. In vitro effects of paclitaxel in 4 ER-positive and 3 ER-negative breast cancer cell lines were investigated by MTT assay. We also investigated members of Bcl-2 family by Western blotting and RT-PCR to clarify their role in paclitaxel resistance both in ER-positive and in ER-negative cells. ER-negative cell lines were more sensitive to paclitaxel than ER-positive cells. ER-negative KPL-4 and ZR-75-30 cells, which were sensitive to paclitaxel, became resistant when they were treated with demethylation agent, 5-aza-2'-deoxycytidine. Analysis of proapoptotic (Bax) and antiapoptotic (Bcl-2) molecules suggested that Bcl-2 is likely to have a role in the resistance of ER-positive cells. Bcl-2 expression was increased in a time-dependent manner after treatment of ER-positive cell lines with estrogen (E2). On the other hand, Bcl-2 was not detected in ER-negative cell lines. However, no significant difference was detected for Bax mRNA levels before and after E2 treatment in ER-positive and negative cell lines. Activation of ER gene expression in ER-negative KPL-4 cells by 5-aza-2'-deoxycytidine resulted in up-regulation of Bcl-2 mRNA. To support our data, we examined paclitaxel sensitivity in ER-negative MDA-MB-231 and ER stable transfectant cells S30 and JM6. This experiment also showed ER-negative cells were sensitive to paclitaxel but ER-positive cells were resistant to it. These results suggest that ER influenced chemosensitivity to paclitaxel through regulation of Bcl-2 family and regulation of the pathway may be crucial to increase the efficacy of taxanes in ER-positive breast cancer.

Background: Monocyte chemotactic protein-1 (MCP-1) is a chemokine ligand that has been associated with aggressive behavior in breast and prostate cancer. The present study was performed to determine if there is a relationship between the expression of MCP-1 in papillary thyroid carcinoma (PTC) and factors indicative of aggressive behavior in this disease., Methods: The subjects of this study were 115 patients with PTC. MCP-1 expression was determined using a semiquantitative scoring system for immunohistochemical staining of MCP-1 in resected PTC samples. There were four levels of immunohistochemical staining intensity, and the population of cells that positively stained for MCP-1 was graded at four levels. The scores for the intensity of immunohistochemical staining for MCP-1 and for the percentage of cells that stained for MCP-1 were used to generate a range from 0 to 9 for scoring MCP-1 expression., Results: Positive staining for MCP-1 was observed in the cytoplasm of PTC cells and stroma cells in 79.2% of the specimens. Expression levels of MCP-1 in PTC cells were positively correlated with tumor size (p < 0.05) and lymph node involvement (p < 0.05). In addition, the expression of MCP-1 in PTC cells level was an independent predictive factor for recurrence of PTC in an analysis that included age, sex, tumor size, extrathyroidal infiltration, and lymph node involvement (p < 0.005)., Conclusions: MCP-1 expression in PTC may stimulate the aggressive behavior of this tumor or it may be a marker for aggressive behavior. Previous reports with non-thyroid tumor cells favor the hypothesis that MCP-1 expression promotes aggressive behavior in PTC.

The intrinsic subtype has demonstrated that breast cancers can be classified into biologically and clinically meaningful subgroups. Most breast tumors categorized as one of the intrinsic subtypes, i.e., basal-like, have an estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative phenotype, so-called triple-negative (TN) phenotype; however, TN breast cancer is not a synonym for basal-like subtype. TN breast cancers account for 10-20% of all breast cancers, and are more biologically aggressive than breast cancers of other subgroups. Tailored therapies, such as endocrine therapy and anti-HER2 therapy, are not applicable to TN breast cancer. To develop novel strategies against TN breast cancer, it is essential to understand the specific pathways driving the aggressive behavior of TN breast cancer. Preclinical and clinical studies have suggested that DNA-damaging agents and poly ADP-ribose polymerase inhibitors are active in TN breast cancer harboring BRCA1 dysfunction; anti-epidermal growth factor receptor (EGFR) antibodies and EGFR tyrosine kinase inhibitors are active in TN breast cancer with EGFR gene amplification; dasatinib is active in TN breast cancer with activated Src tyrosine kinases; inhibitors of a mammalian target of rapamycin are active in TN breast cancer with loss of PTEN tumor suppressor; antiangiogenic therapies enhance antitumor activity of chemotherapeutic agents in hypervascular TN breast cancer; and irinotecan, trabectedin, ixabepilone, and ABI-007 are active in TN breast cancer. A number of clinical trials are ongoing to clarify the antitumor activity of these challenging treatment strategies. Further biological characterization of TN breast cancer is needed to develop more specific treatment strategies against TN breast cancer.

We experienced a case of paclitaxel- and trastuzumab-resistant recurrent breast cancer with liver metastases showing significant improvement by S-1. A 76-year-old woman was diagnosed with left breast cancer (T2N1M0, Stage II B). She received total mastectomy and CEF (cyclophosphamide 500 mg/m(2), epirubicin 60 mg/m(2), 5-FU 750 mg/m(2)) as adjuvant chemotherapy in March 2004. But twelve months later, she was referred to our clinic for management of lung and left supraclavicular lymph node metastases. Then weekly paclitaxel (80 mg/m(2)) and trastuzumab were started. After 2 cycles of weekly paclitaxel and trastuzumab treatment, lung and lymph node metastases were reduced and the patient showed a clinical response (CR), so she was treated by trastuzumab only. But seven months later, she was diagnosed as a recurrence of liver metastases. She was treated by combined paclitaxel and trastuzumab again, but liver metastases and tumor marker were progressive. S-1 was administered orally 100 mg/day every day for 4 weeks, followed by a 2-week rest interval as 1 cycle, and trastuzumab was injected at 2 mg/kg/week for every weeks. After 2 courses of the treatment, the level of tumor marker and tumor size of liver metastases were reduced. Only rash (grade 1) was observed during treatment. The treatment of S-1 is thought to be effective for taxane-resistant recurrent breast cancer.

Epirubicin exerts its anti-tumor effect through binding to topoisomerase IIalpha (TOP2A) and inducing DNA double-strand breaks. BRCA1 is involved in the repair of these breaks. We investigated the relationship between TOP2A or BRCA1 immunohistochemical expression and pathological response in 108 primary breast cancers treated with epirubicin-based regimens. The pCR (pathological complete response) rate for TOP2A-positive (17%) was significantly (P < 0.005) higher than for TOP2A-negative (2%), while the pCR rate for BRCA1-negative (11%) was non-significantly higher than for BRCA1-positive (5%). The pCR rate of TOP2A-positive and BRCA1-negative (30%) was significantly higher than for TOP2A-negative and BRCA1-positive (3%; P < 0.05), or TOP2A-negative and BRCA1-negative (0%; P < 0.005). The TOP2A-positive and BRCA1-negative phenotype associates with a favorable response to epirubicin-based regimens.

Purpose: Predictive factors for response to docetaxel in human breast cancers have yet to be identified. The aim of the present study was to investigate the relationship of various clinicopathological and biological parameters with pathological response to docetaxel in the neoadjuvant setting., Methods: The study population comprised 78 patients with primary breast cancers who were treated with docetaxel [60 mg/m(2); four (median) cycles, range 3-6; q3w] as neoadjuvant therapy and subsequently treated with mastectomy or breast conserving surgery. Tumor samples obtained before chemotherapy were subjected to histological examination and immunohistochemistry of HER-2 and Ki-67., Results: The pathological complete response (pCR) rate was significantly (P=0.04) higher for tumors with low nuclear grade (NG-I or -II) (21%) than for tumors with high NG (NG-III) (5%). The pCR rate (20%) of small (5 cm) tumors (5%). Combined analysis of NG and tumor size showed that low-NG small tumors have a higher response rate (30%) than high-NG small tumors (11%; P=0.13), low-NG large tumors (11%; P=0.15), and high-NG large tumors (0%; P=0.009). No statistically significant association was observed between pCR rate and menopausal status, lymph node status, ER, PR, HER-2, or Ki-67., Conclusions: Low nuclear grade, but not cell proliferation determined by Ki-67, is associated with a good pathological response to docetaxel. Combination of low nuclear grade and small tumor size may be useful for the selection of breast tumors with a high pCR rate (30%).

Background: The combination of capecitabine and paclitaxel (XP) has demonstrated synergistic antitumor activity in preclinical models. Three-weekly XP regimens have demonstrated excellent efficacy in phase II and III trials in metastatic breast cancer. We conducted a dose-finding study to identify the recommended 4-weekly XP regimen in patients with inoperable or recurrent breast cancer for phase II evaluation., Methods: Eligible patients had inoperable or recurrent breast cancer previously treated with chemotherapy (but not capecitabine or paclitaxel) in the (neo)adjuvant or metastatic setting. Each 4-week treatment cycle consisted of escalating doses of capecitabine (628 or 829 mg/m(2) twice daily [b.i.d.] on days 1-21) and paclitaxel (80 or 90 mg/m(2) on days 1, 8, and 15). Dose-limiting toxicities (DLT) were evaluated during the first two cycles., Results: Nine patients were treated. At dose level 1 (capecitabine 628 mg/m(2) b.i.d. plus paclitaxel 80 mg/m(2)), one patient experienced a DLT (grade 3 non-hematologic toxicity). There were no further DLTs at dose level 1 or 2. Although the MTD was not reached, dose level 2 (capecitabine 829 mg/m(2) b.i.d., days 1-21, plus paclitaxel 80 mg/m(2), days 1, 8, and 15, every 28 days) is recommended for phase II evaluation, taking into consideration the single-agent doses used in Japan and the doses identified in Western studies of 3-weekly XP. The overall response rate was 44%; all patients treated at dose level 2 achieved a partial response., Conclusions: This 4-weekly XP regimen was well tolerated, active in patients with pretreated advanced breast cancer, and could be given as outpatient treatment. These results are consistent with findings of phase II and III trials evaluating 3-weekly regimens, and indicate that further investigation of a 4-weekly XP regimen is warranted.

Breast cancer most frequently causes bone metastases in solid tumors. It has been known that there is a vicious cycle consisting of tumor cells, osteoblasts, osteoclasts and various humoral factors in osteolytic lesions. Although systemic therapy is a main treatment of bone metastases, local therapies, such as radiotherapy and surgical therapy, are also promptly needed when bone-related complications occur. In recent years, anti-osteoclast agents, bisphosphonates significantly contribute to the delay of occurrence of bone-related complications. Postoperative adjuvant therapy significantly reduces the incidence of recurrence in breast cancer patients. Chemotherapy and LH-RH agonists cause ovarian function suppression in premenopausal patients, and aromatase inhibitors cause estrogen deprivation in postmenopausal patients. These effects cause unbalance of bone metabolism, loss of bone density and increase in the incidence of fractures. Improvement of these bone-related adverse effects and careful follow-ups are needed for breast cancer patients.

Background: There are three third-generation aromatase inhibitors (AI) available in Japan. Though it is supposed that they can be administered sequentially because of their little cross-resistant effect, it is not definite which sequential treatment is best., Purpose: To examine retrospectively the difference in efficacy by the AI sequence when anastrozole ( ANA) and exemestane (EXE) are clinically administered sequentially for patients with metastatic breast cancer., Patients: We examined 22 patients with metastatic breast cancer administered ANA alone as first-line AI treatment, EXE alone as second-line (A--> E group), and 13 patients given EXE alone as first-line AI treatment (E --> A group) since December 2002 in our hospital., Method: In the A --> E and E --> A group, we examined overall response rate, clinical benefit (CB) rate, time to progression (TTP) for the first- and second-line treatment, respectively, overall survival (OS) after starting AI and successive efficacy of the two AIs., Results: There were no significant differences between the two groups in patient characteristics and history of prior treatments. Overall response rate of the first-line treatment in the A--> E and the E--> A group was 31.8% and 38.5%, and the CB rate was 68.2% and 53.8%, respectively. Overall response rate of the second-line treatment in the A --> E (22 cases) and the E --> A (3 cases) group was 13.6% and 0%, and the CB rate was 36.4% and 33.3%, respectively, reflecting no particular differences. In the A --> E group, five (33.3%) among 15 cases obtained CB by EXE and three (42.6%) among 7 cases who did not obtain CB by ANA did so by EXE. Also, in the E--> A group, one among 3 cases who did not obtain CB by EXE did so by ANA. No significant differences were observed in TTP and OS between the two groups., Conclusion: When ANA and EXE are administered sequentially, no particular difference in efficacy occurs due to the sequence. In some cases, 2 agents become effective successively, and cases resistant to first-line treatment can expect efficacy from second-line treatment. Sequential treatment with the two agents seems to provide very significant efficacy in clinical practice regardless of the order of administration.

Purpose of Review: Current topics on adjuvant therapy for premenopausal patients with breast cancer are reviewed., Recent Findings: The Early Breast Cancer Trialists' Collaborative Group overview showed that women under 50 received a remarkable benefit from postoperative chemotherapy and endocrine therapy. The impact of chemotherapy-induced amenorrhea on patient outcome remains to be defined. The addition of luteinizing-hormone-releasing hormone agonists to tamoxifen, chemotherapy or both significantly reduced both recurrence and death in premenopausal patients with endocrine-responsive breast cancer. Exploratory trials are investigating the use of more complete estrogen suppression with luteinizing-hormone-releasing hormone agonists with aromatase inhibitors. There is no consensus on how long these agonists should be used in the adjuvant setting. Chemotherapy-induced ovarian failure is frequently associated with infertility. The suppression of ovarian function using luteinizing-hormone-releasing hormone agonists during chemotherapy seems promising for preserving ovarian function. Chemotherapy-induced ovarian failure and prolonged luteinizing-hormone-releasing hormone agonist therapy cause premature menopausal symptoms. To minimize the menopausal symptoms, intervention strategies should be investigated., Summary: There are a number of unresolved issues in terms of the optimal use of chemotherapy and endocrine therapy in premenopausal patients with breast cancer. Well-designed and well-organized randomized clinical trials are essential to resolve these issues.

A recent report indicated that a high prevalence of basal-like breast tumors (estrogen receptor [ER]-negative, progesterone receptor [PR]-negative, human epidermal growth factor receptor [HER] 2-negative, and cytokeratin 5/6-positive and/or HER1-positive) could contribute to a poor prognosis in African American women with breast cancer. It has been reported that Japanese women with breast cancer have a significantly better survival rate than other races in the USA. These findings suggest that breast cancers in Japanese women have favorable biological characteristics. To clarify this hypothesis, we conducted a cohort study to investigate the prevalence of intrinsic subtypes and prognosis for each subtype in 793 Japanese patients. This study revealed a very low prevalence (only 8%) of basal-like breast tumors with aggressive biological characteristics in Japanese patients. Survival analysis showed a significantly poorer prognosis in patients with basal-like tumors than in those with luminal A tumors (ER- and/or PR-positive, and HER2-negative) with favorable biological characteristics. These findings support the hypothesis that breast cancers in Japanese women have more favorable biological characteristics and a better prognosis than those in other races. In conclusion, the prevalence of basal-like breast tumors could influence the prognosis of breast cancer patients of different races. The prevalence of intrinsic subtypes should be taken into account when analyzing survival data in a multi-racial/international clinical study.

BRAF (7q24) encodes a serine/threonine protein kinase, and its expression level varies in different tissues. Although a high prevalence of BRAF mutation has been suggested as an important event in thyroid tumorigenesis, little is known about the expression pattern of B-Raf in the thyroid. Thus, we examined the expression of B-Raf in various neoplastic and nonneoplastic thyroid tissues and compared it with BRAF mutational status. Normal and hyperplastic thyroid tissues showed focal and faint immunoreactivity for B-Raf, especially in cuboidal follicular cells of small follicles. In contrast, diffuse expression of B-Raf was observed in follicular adenomas and well-differentiated carcinomas. The missense point mutation BRAF(V600E) was identified in 42% (13/31 cases) of papillary carcinomas and 33% (5/15 cases) of undifferentiated carcinomas but not in normal thyroid tissues, nodular hyperplasia, follicular adenomas, or follicular carcinomas. The immunohistochemical expression of B-Raf did not correlate with BRAF mutational status. Moreover, Western blotting revealed that B-Raf expression in thyroid carcinoma cell lines was also independent of BRAF mutation. Serum or fibroblast growth factor-1 stimulation further activates ERK1/2 in heterozygous BRAF(V600E)-positive carcinoma cells as well as BRAF(V600E) mutation-negative carcinoma cells. In conclusion, heterogeneous focal expression of wild-type B-Raf in nonneoplastic tissues may play a role in the growth or functional activity of thyroid follicular cells. In contrast, diffuse expression of wild-type and/or mutant B-Raf may be involved in the tumorigenic process resulting in activation of the mitogen-activated protein kinase signaling pathway in cooperation with other genetic abnormalities and activation of ligand-receptor signaling pathways.

Fibroblast growth factor (FGF) signals play fundamental roles in development and tumorigenesis. Thyroid cancer is an example of a tumor with nonoverlapping genetic mutations that up-regulate mitogen-activated protein kinase (MAPK). Here, we show that FGF receptor 1 (FGFR1), which is expressed mainly in neoplastic thyroid cells, propagates MAPK activation and promotes tumor progression. In contrast, FGFR2 is down-regulated in neoplastic thyroid cells through DNA promoter methylation. Reexpression of FGFR2 competes with FGFR1 for the immediate substrate FGFR substrate 2 to impede signaling upstream of the BRAF/MAPK pathway. These data unmask an epigenetically controlled FGFR2 signal that imposes precisely on the intragenically modified BRAF/MAPK pathway to modulate thyroid cancer behavior.

We report three radioiodine-resistant patients with metastatic papillary thyroid carcinoma administered docetaxel. Patient 1: Bi-weekly docetaxel was administered to a 67-year-old woman with clavicle, cervical lymph node and lung metastases that had progressed after external irradiation and radioiodine therapy. Stable disease was maintained for 18 months without elevation of serum thyroglobulin. Patient 2: Bi-weekly docetaxel was administered to a 72-year-old man with lung metastases that had progressed after radioiodine therapy. Stable disease was maintained for 14 months. Patient 3: Bi-weekly docetaxel was administered to a 58-year-old woman with lung metastases that had progressed after radioiodine therapy. Stable disease was maintained for 18 months with slight reduction of serum thyroglobulin. In all three patients, doubling time of tumor growth was revealed to be far prolonged after docetaxel administration without distinct adverse events. Since no effective systemic treatment has been established for radioiodine-resistant patients with metastatic papillary thyroid carcinoma,docetaxel therapy was supposed to be a viable alternative for them.

We recently reported that heparanase, one of the extracellular matrix-degrading enzymes, which plays a critical role in cancer progression, is located not only in the cytoplasm but also in the nucleus. Here we identified nuclear translocation of heparanase as a key step in cell differentiation. We applied an in vitro differentiation model of HL-60 cells with 12-0-tetradecanoylphorbol-13-acetate (TPA), in which nuclear translocation of heparanase was observed using immunohistochemical analysis. In this system, nuclear translocation of heparanase was abolished by inhibitors of heat shock protein 90 (HSP90), suggesting the involvement of HSP90 in translocation of heparanase. We further confirmed that overexpression of active form of heparanase induced differentiation of HL-60 cells, although the catalytic negative form of heparanase did not. Therefore we speculate that nuclear translocation of enzymatically active heparanase may be involved in cellular differentiation. Our results suggest that a novel function of heparanase upon cell differentiation would raise a potential new strategy for cancer therapy of promyeloid leukemia and other types of cancer.