Seishi Kono

Absence of primary cilia in cell cycle-arrested human breast cancer cells

Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki‐67, primary cilia were present only in 20–40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indicate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells.

Predictive factors for anthracycline-based chemotherapy for human breast cancer

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.

Immunohistochemical study of the relationship between Ki-67 labeling index of proliferating cells of gynecomastia, histological phase and duration of disease.

Gynecomastia is a benign proliferative lesion caused by various etiological factors and may result from a relative imbalance between serum estrogen and androgen levels. The histological alterations are similar, and gynecomastia can progress from a florid type to a fibrous type. The Ki‐67 labeling index (LI) of gynecomastia specimen was investigated and higher Ki‐67 LI was observed in florid and intermediate than in fibrous gynecomastia (P = 0.017). A correlation was found between the duration of disease and Ki‐67 LI (P = 0.041): the shorter the duration the higher the Ki‐67 LI. Thus, Ki‐67 LI seems a useful tool to examine proliferation activity of gynecomastia and can assist in determination of appropriate treatment of gynecomastia with hormonal therapy.

Differences in elongation of very long chain fatty acids and fatty acid metabolism between triple-negative and hormone receptor-positive breast cancer

BackgroundTriple-negative breast cancer (TN) is more aggressive than other subtypes of breast cancer and has a lower survival rate. Furthermore, detailed biological information about the disease is lacking. This study investigated characteristics of metabolic pathways in TN.MethodsWe performed the metabolome analysis of 74 breast cancer tissues and the corresponding normal breast tissues using LC/MS. Furthermore, we classified the breast cancer tissues into ER-positive, PgR-positive, HER2-negative breast cancer (EP+H-) and TN, and then the differences in their metabolic pathways were investigated. The RT-PCR and immunostaining were carried out to examine the expression of ELOVL1, 2, 3, 4, 5, 6, and 7.ResultsWe identified 142 of hydrophilic metabolites and 278 of hydrophobic lipid metabolites in breast tissues. We found the differences between breast cancer and normal breast tissues in choline metabolism, glutamine metabolism, lipid metabolism, and so on. Most characteristic of comparison between EP+H- and TN were differences in fatty acid metabolism was which were related to the elongation of very long chain fatty acids were detected between TN and EP+H-. Real-time RT-PCR showed that the mRNA expression levels of ELOVL1, 5, and 6 were significantly upregulated by 8.5-, 4.6- and 7.0-fold, respectively, in the TN tumors compared with their levels in the corresponding normal breast tissue samples. Similarly, the mRNA expression levels of ELOVL1, 5, and 6 were also significantly higher in the EP+H- tissues than in the corresponding normal breast tissues (by 4.9-, 3.4-, and 2.1-fold, respectively). The mRNA expression level of ELOVL6 was 2.6-fold higher in the TN tumors than in the EP+H- tumors. During immunostaining, the TN and EP+H- tumors demonstrated stronger ELOVL1 and 6 staining than the corresponding normal breast tissues, but ELOVL5 was not stained strongly in the TN or EP+H- tumors. Furthermore, the TN tumors exhibited stronger ELOVL1 and 6 staining than the EP+H- tumors.ConclusionsMarked differences in fatty acid metabolism pathways, including those related to ELOVL1 and 6, were detected between TN and EP+H-, and it was suggested that ELOVL1 and 6-related fatty acid metabolism pathways may be targets for therapies against TN.

Effectiveness of Pertuzumab, Trastuzumab, and Docetaxel Combination Neoadjuvant Chemotherapy for HER2-Positive Inflammatory Breast Cancer: A Case Report

Background: Inflammatory breast cancer (IBC) is the most aggressive form of primary breast cancer. Case Report: A 40-year-old woman was referred to our hospital for evaluation of an induration in the right breast, suspected to be breast cancer. The tumor was diagnosed as estrogen receptor-negative, progesterone receptor-negative, HER2-positive, T4dN3cM0 stage IIIc IBC with axillary lymph node metastasis. Rather than surgical intervention, we chose a systemic treatment approach with pertuzumab, trastuzumab, and docetaxel (PTD) combination therapy which was shown to be effective for HER2-positive IBC in the NeoSphere trial. After 4 cycles of treatment, the patient had a partial response, allowing mastectomy of the right breast and axillary lymph node dissection to achieve local control. We review this case because of the success of PTD combination neoadjuvant chemotherapy for HER2-positive IBC. Conclusion: To improve the poor prognosis of IBC, combined modality therapy is required, including chemotherapy and local treatment such as surgery and/or radiation therapy. In this case, combination neoadjuvant chemotherapy with PTD for HER2-positive IBC was effective, and this regimen may contribute to further improvements in the cure rate for this malignancy.

Abstract 319: Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer

While it is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs, three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The aim of this study was to reveal differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We first evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status, and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474, and T-47D) developed dense multi-cellular spheroids (MCSs) in 3D-culture, and tended to show greater resistance to paclitaxel and doxorubicin than in 2D-culture. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB-231) developed only loose MCSs in 3D-culture, and showed drug sensitivities similar to those in 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved PARP expression in 2D- than in 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected cells from paclitaxel-induced apoptosis. Hypoxia, as measured with phosphorescent LOX-1, was observed only in dense 3D-MCSs. BT-549 had considerably fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0 dormant sub-population may be responsible for its drug resistance in 3D-culture. In addition, BT-474 had a lower level of caspase-3 in 3D- than in 2D-culture, suggesting that the 3D-environment was anti-apoptotic. We next examined whether these findings were reproduced in primary cells utilizing a breast cancer patient-derived xenograft (PDX). The fresh disaggregated PDX tissue developed dense MCSs in 3D-culture. We compared staining for Ki-67, and caspase-3 and -8 in 2D- and 3D-cultured primary cells obtained from the PDX, and the original patient tumor (Ki-67 only): 2D-cultured cells showed much greater proportions of Ki-67-positive and caspase-3-positive cells than the others. 3D-cultured cells tended to show greater resistance to paclitaxel and doxorubicin compared to the others. Residual spheroids after exposure to paclitaxel did not grow after removal of the drug but majority of cells in the spheroids appeared alive based on their excluding trypan blue dye. In the residual spheroids, greater population of cells showed phosphorylated histone H2AX expression than those in the spheroids unexposed to paclitaxel, suggesting that chemotherapy-induced senescence associated with drug resistance. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important characteristics of tumor grown in vivo, namely hypoxia, dormancy, anti-apoptotic features, and their resulting drug resistance. 3D primary culture will be a model for the study of chemotherapy-induced senescence. Citation Format: Yoshinori Imamura, Toru Mukohara, Yohei Shimono, Yohei Funakoshi, Naoko Chayahara, Masanori Toyoda, Naomi Kiyota, Shintaro Takao, Seishi Kono, Tetsuya Nakatsura, Hironobu Minami. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 319. doi:10.1158/1538-7445.AM2015-319

Adipose-derived stem cells enhance human breast cancer growth and cancer stem cell-like properties through adipsin

Tumor microenvironment plays a key role for tumor development and progression. Although adipose tissue is a predominant component of stroma in mammary tissues and secretes various cytokines, chemokines and growth factors, roles of adipocytes in breast cancers remain to be elucidated. In this study, we found that adipsin, an adipokine secreted from mammary adipose tissues, enhanced proliferation and cancer stem cell (CSC)-like properties of human breast cancer patient-derived xenograft (PDX) cells. Adipsin was predominantly expressed in both adipose tissues of the surgical specimens of breast cancer patients and adipose-derived stem cells (ADSCs) isolated from them, and its expression level was significantly higher in obese patients. ADSCs significantly enhanced the sphere-forming ability of breast cancer PDX cells derived from both estrogen receptor-positive and -negative breast cancer PDX cells. Suppression of adipsin-mediated signaling by a specific inhibitor or adipsin knockdown in ADSCs significantly decreased the sphere-forming ability and the expression of CSC markers in co-cultured breast cancer PDX cells. Growth of breast cancer PDX tumors was significantly enhanced by co-transplantation with ADSCs in vivo, and it was weakened when co-transplanted with the adipsin knocked-down ADSCs. These results suggest that adipsin is an important adipokine secreted from mammary adipose tissue that functions as a component of tumor microenvironment and a CSC niche in breast cancers.

Expression of programmed death-1 in sentinel lymph nodes of breast cancer

To explore whether lymphocytes in sentinel lymph nodes (SLNs) are highly exposed to tumor neoantigens and thus express high level of programmed death 1 (PD‐1), we examined PD‐1 expression in SLNs and non‐sentinel regional lymph nodes (non‐SLNs) in breast cancer.

Improvement of psoriasiform eruptions after resection of breast cancer

EJD, vol. 27, n◦ 6, November-December 2017 possibly resulting in more vegetative lesions, responsible for the white areas seen on dermoscopy. The current case report highlights the typical dermoscopic characteristics of acantholytic dermatoses together with their common histological origin. It also underlines the usefulness of dermoscopy in the diagnosis of the rare disease, Hailey-Hailey, that may often remain undiagnosed, especially if it runs an indolent course and responds to topical steroids and anti-fungal agents.

Abstract P1-12-04: Factors influencing on discontinuation of adjuvant anastrozole in postmenopausal Japanese breast cancer patients: Results from a prospective multicenter cohort study of patient-reported outcomes

Background: Adjuvant five-year treatment with aromatase inhibitors is standard for postmenopausal women with estrogen receptor positive breast cancer. However, aromatase inhibitor-related adverse events including joint symptoms and vasomotor symptoms have a strong impact on patients9 quality of life and sometimes result in treatment discontinuation. The aim of this study is to determine risk factors for discontinuation of endocrine therapy in Japanese postmenopausal breast cancer patients treated with adjuvant anastrozole in a prospective cohort study based on patient-reported outcomes (PROs). Patients and Methods: A total of 391 postmenopausal Japanese women with estrogen receptor-positive breast cancer and treated with adjuvant anastrozole were enrolled from 28 centers in this prospective cohort study (SAVS-JP, UMIN000002455). PROs assessment was obtained at baseline, 3, 6, 9 and 12 months which included joint and vasomotor symptoms. Long-term adherence of anastrozole was obtained form 364 out of 391 patients (median follow-up: 44 months, range: 5-105months). We analyzed the relationship of discontinuation of anastrozole with joint and vasomotor symptoms induced by treatment, and patients’ characteristics. Results: Among 364 patients, 64 (17.6%) discontinued, 297 (81.6%) are ongoing and 3 (0.8%) have completed five-year anastrozole treatment. The reasons for discontinuation were recurrence: 20 (31.3%), secondary malignancies: 5 (7.8%), death from non-breast cancer: 1 (1.6%) and adverse events: 38 (59.4%). These 38 patients who stopped treatment caused by adverse events were compared with other 323 patients. Joint and vasomotor symptoms were categorized into grade 0 (no symptom or no change from baseline), grade 1+2 (mild+moderate) and grade 3 (severe). Grades of joint symptoms were significantly associated with discontinuation of anastrozole (Grade 0: 9.7%, grade 1+2: 7.8%, grade 3: 25.0%, p=0.02). Patients with longer time after menopause (16 years or longer) were significantly higher frequency of discontinuation as compared with shorter time after menopause (0-15years) (14.9% vs 8.0%, p=0.04). Univariate analysis revealed that grade 3 joint symptoms (odds ratio: 3.67, 95% confidence interval: 1.34-10.04, p=0.01) and longer time after menopause (OR: 2.01, 95%CI: 1.01-4.00, p=0.04) were significant risk factors for discontinuation. By multivariate analysis, both grade 3 joint symptoms and long time after menopause were independently associated with discontinuation. Conclusion: In the present study, we have identified that grade 3 joint symptoms and longer time after menopause were risk factors for discontinuation of adjuvant anastrozole. These data might give us useful information for counseling in patients with adjuvant aromatase inhibitors for postmenopausal Japanese women. Citation Format: Chiyomi Egawa, Shintaro Takao, Kazuhiko Yamagami, Masaru Miyashita, Masashi Baba, Shigetoshi Ichii, Muneharu Konishi, Yuichiro Kikawa, Junya Minohata, Toshitaka Okuno, Keisuke Miyauchi, Kazuyuki Wakita, Hirofumi Suwa, Takashi Hashimoto, Masayuki Nishino, Takashi Matsumoto, Toshiharu Hidaka, Yutaka Konishi, Yoko Sakoda, Akihiro Miya, Masahiro Kishimoto, Hidefumi Nishikawa, Seishi Kono, Ikuo Kokufu, Isao Sakita, Koushiro Kitatsuji, Koushi Oh, Yasuo Miyoshi. Factors influencing on discontinuation of adjuvant anastrozole in postmenopausal Japanese breast cancer patients: Results from a prospective multicenter cohort study of patient-reported outcomes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-04.