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Background: Some germline pathogenic variants (PVs) have been observed with high frequency in certain regions and races and are called founder mutations. Founder mutations in BRCA1/2 genes have rarely been reported in Japan. At Kakogawa Central City Hospital, BRCA2 c.5576_5579del accounted for approximately 70% of PVs detected by BRCA1/2 genetic testing. Therefore, we conducted a retrospective observational study to investigate its distribution in Hyogo Prefecture. Patients and Methods: This study was conducted at Kobe University Hospital and three collaborating institutions. Breast cancer patients with germline PVs detected by BRCA1/2 genetic testing between July 1, 2018 and March 31, 2021 were included. The clinical characteristics and breast cancer subtypes were compared between carriers of BRCA2c.5576_5579del and those of other PVs. The detection rate of BRCA2c.5576_5579del was also compared with that observed in a previous report. Results: A total of 38 breast cancer patients were included; 14 of them underwent testing for companion diagnosis to poly ADP-ribose polymerase inhibitor treatment and 24 underwent testing for diagnosis of hereditary breast and ovarian cancer. PVs in BRCA1 andBRCA2 were detected in 12 and 26 patients, respectively, 12 of whom were BRCA2c.5576_5579del carriers. There were no clinicopathological differences between BRCA2PV carriers and BRCA2 c.5576_5579del carriers. BRCA2c.5576_5579del accounted for 30.8% of the PVs detected, with a particularly high frequency of 72.7% at Kakogawa Central City Hospital. Conclusion: Our study revealed a strong founder effect for BRCA2 c.5576_5579del in breast cancer patients in Hyogo Prefecture, especially in Kakogawa City. In the future, a survey of the distribution of the BRCA2 c.5576_5579del carriers may provide more clarity regarding their localization.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44+ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell–related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers., Members of the S100 protein family, including S100A10, were highly upregulated in metastasized cancer stem cells (CSCs) compared with primary CSCs. Among them, S100A10 functioned as an enhancer of both CSC properties and invasion abilities, and its knockdown suppressed liver metastases in a mouse xenograft model.

Background Five-year adjuvant treatment with aromatase inhibitors is a standard treatment for postmenopausal women with estrogen receptor-positive breast cancer. However, aromatase inhibitor-related adverse events, including joint and vasomotor symptoms, have a strong impact on patient quality of life and sometimes cause the discontinuation of treatment. The aim of this prospective cohort study based on patient-reported outcomes (PROs) was to examine the influence of adverse events on prognosis in Japanese postmenopausal patients with breast cancer treated with adjuvant anastrozole. Patients and Methods A total of 391 postmenopausal Japanese women with estrogen receptor-positive breast cancer who were treated with adjuvant anastrozole were enrolled from 28 centers in this prospective cohort study (SAVS-JP, UMIN000002455). PRO assessments of adverse events, which included joint and vasomotor symptoms, were obtained at baseline and 3, 6, 9, and 12 months. Symptoms were assessed and assigned to one of four categories: none (G0), mild (G1), moderate (G2), and severe (G3). The questionnaires covered joint symptoms (arthralgia, decrease in range of joint motion, and joint stiffness), vasomotor symptoms (hot flashes, night sweats, and cold sweats), and adherence to anastrozole. The median follow-up was 82 months, and we analyzed the prognosis in patients, focusing on the occurrence of joint and vasomotor symptoms induced by treatment. Results Of the 391 patients, 204 (52.2%) completed 5-year treatment, 75 (19.2%) discontinued (adverse events (n=35), recurrence (n=19), secondary malignancies (n=4), death from non-breast cancer (n=4), and other reasons (n=13)), 48 (12.3%) are receiving treatment, and 64 (16.4%) were unknown. Patients who experienced G2+3 joint symptoms had significantly better disease-free survival (DFS) compared with patients with G0+1 joint symptoms (5-year DFS, 96.1% vs. 83.3%, p=0.002). Similarly, the DFS in patients with G2+3 vasomotor symptoms was significantly better than that in those with G0+1 vasomotor symptoms (5-year DFS, 93.5% vs. 86.3%, p=0.049). Joint symptoms were marginally associated with overall survival (OS) (p=0.062), but no significant association was found with vasomotor symptoms. The DFS was superior in patients with G2+3 joint and vasomotor symptoms (5-year DFS, 100%), inferior in those with G0+1 joint and vasomotor symptoms (5-year DFS, 82.6%), and intermediate in others (5-year DFS, 93.3%; p=0.0024). By multivariable analysis including tumor size and grade, G2+3 joint symptoms were a significant and independent predictive factor for DFS (hazard ratio, 0.298, 95% confidence interval 0.122-0.621, p=0.0007). Conclusion Although adverse events might make patients uncomfortable and reduce their quality of life, it is suggested that worse or new vasomotor symptoms and severe joint symptoms, especially if they are grade 2 or 3, seem to associate with better prognosis. Citation Format: Chiyomi Egawa, Shintaro Takao, Kazuhiro 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. Influence of aromatase inhibitor-related adverse events on the prognosis in postmenopausal Japanese patients with breast cancer: A prospective multicenter cohort study on patient-reported outcomes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-12-07.

To examine the association of physical activity (PA) with bone health among patients with breast cancer receiving adjuvant aromatase inhibitor (AI) treatment. In this single-center observational study, we enrolled postmenopausal women with primary hormone receptor-positive breast cancer who were receiving adjuvant AI treatment. We assessed patient bone health [bone mineral density (BMD) and biomarkers of bone turnover] as main outcomes. PA was assessed using Baecke physical activity questionnaires (BPAQ) and an accelerometer. Multiple regression analysis was performed after adjustment for age, body mass index, smoking history and duration of AI treatment. For missing data, multiple imputation analysis was adapted. The mean age of the 53 enrolled patients was 67.4 ± 8.4 years. The mean duration of AI administration was 25.7 ± 18.9 months. The most frequently administered AI was anastrozole (73.6%). Although not related to BMD, PA was related to bone turnover. Serum collagen type I amino-terminal propeptide, a bone formation marker, was associated with only light PA (t = − 2.55, p = 0.015), while tartrate-resistant acid phosphatase 5b, a bone absorption marker, was associated with work index in the BPAQ subscale and light PA (t = − 2.28, p = 0.028, t = − 2.26, p = 0.031, respectively). The results for all patients were similar to those observed in the multiple imputation analysis. PA was significantly associated with bone turnover among cancer patients receiving AI treatment. Light PA and PA in the work domain were the most important factors among various PA intensities and PA domains.

Simple Summary Obesity, which is characterized by the excess of adipose tissue, is associated with an increased risk of multiple cancers. We have previously reported that adipsin, a secreted factor from adipocytes, enhances cancer cell proliferation and stem cell properties. In this study, we found that adipsin affected adipocytes themselves and enhanced their secretion of hepatocyte growth factor (HGF). We found that HGF enhanced the adipocyte-cancer cell interactions as a downstream effector of adipsin. Understanding the adipocyte-cancer cell interaction will provide a novel strategy to treat cancers whose initiation, invasion, and metastatic progression are associated with adipose tissues. Abstract Adipose tissue is a component of the tumor microenvironment and is involved in tumor progression. We have previously shown that adipokine adipsin (CFD) functions as an enhancer of tumor proliferation and cancer stem cell (CSC) properties in breast cancers. We established the Cfd-knockout (KO) mice and the mammary adipose tissue-derived stem cells (mADSCs) from them. Cfd-KO in mADSCs significantly reduced their ability to enhance tumorsphere formation of breast cancer patient-derived xenograft (PDX) cells, which was restored by the addition of Cfd in the culture medium. Hepatocyte growth factor (HGF) was expressed and secreted from mADSCs in a Cfd-dependent manner. HGF rescued the reduced ability of Cfd-KO mADSCs to promote tumorsphere formation in vitro and tumor formation in vivo by breast cancer PDX cells. These results suggest that HGF is a downstream effector of Cfd in mADSCs that enhances the CSC properties in breast cancers.

Adverse events related to endocrine therapies have a major impact not only on patients’ quality of life but also on treatment discontinuation. Although vasomotor symptoms induced by aromatase inhibitors are frequently recognized, risk factors, especially for Japanese women, are not well reported. To identify risk factors for vasomotor symptoms of Japanese breast cancer patients treated with adjuvant anastrozole, we conducted a prospective cohort study based on patient-reported outcomes (PROs). For this prospective cohort study (SAVS-JP, UMIN000002455), 391 postmenopausal Japanese estrogen receptor-positive breast cancer patients who were treated with adjuvant anastrozole were recruited from 28 centers. The PRO assessment was obtained from a self-reported questionnaire at baseline, 3, 6, 9 and 12 months between August 2009 and April 2012. Vasomotor symptoms, comprising hot flashes, night sweats, and cold sweats, were categorized into four grades (none, Grade 1: mild, Grade 2: moderate, Grade 3: severe). Pre-existing symptoms were only included if they had become worse than at baseline. Hot flashes, night sweats, and cold sweats at baseline were reported by 20.5, 15.1, and 8.2 % of the patients, respectively, and new appearance or worsening of symptoms in comparison with baseline by 38.4, 29.3, and 28.7 %, respectively. About 80 % of newly occurring symptoms were Grade 1, and less than 5 % were Grade 3. Vasomotor symptoms were reported by 201 out of 362 patients (55.5 %) during the first year and the mean time to onset was 5.6 months. Patients with vasomotor symptoms were significantly younger (mean 62.8 years, range 38–86 vs 64.7 years, range 37–84; p = 0.02), had higher body mass index (BMI) (23.4 kg/m2, range 15.8–39.9 vs 22.4 kg/m2, range 15.8–34.9; p = 0.01), had vasomotor symptoms sooner after menopause (12.4 years, range 0–51 vs 15.1 years, range 1–37; p = 0.002), and had more menopausal disorders during menopause (63.3 vs 36.7 %; p = 0.002). Multivariate analysis showed that BMI [odds ratio (OR) 1.09 per unit of increase, 95 % confidence interval (CI) 1.02–1.16; p = 0.009] and experiencing menopausal disorders (OR 2.11, 95 % CI 1.35–3.30; p = 0.001) were significantly associated with vasomotor symptoms. High BMI and experiencing menopausal disorders at menopause were found to be significantly associated with the occurrence of vasomotor symptoms. These findings are expected to prove useful for the management of postmenopausal Japanese women treated with aromatase inhibitors.

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 patients' 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.

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; therefore three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The purpose of this study was to reveal the differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We 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 multicellular spheroids (MCSs) in 3D-culture, and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cells. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB‑231) developed only loose MCSs in 3D, and showed drug sensitivities similar to those found in the 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved-PARP expression in the 2D-culture compared with the 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected the cells from paclitaxel-induced apoptosis. Hypoxia was observed only in the dense 3D-MCSs. BT-549 had fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0-dormant subpopulation was responsible for its drug resistance in the 3D-culture. BT-474 had a lower level of caspase-3 in the 3D- than in the 2D-culture, suggesting that the 3D-environment was anti-apoptotic. Finally, we compared staining for Ki-67 and caspases in the 2D- and 3D-primary‑cultured cells originating from a patient-derived xenograft (PDX), fresh PDX tumor, and the patient's original tumor; 2D-cultured cells showed greater proportions of Ki-67-positive and caspase-3-positive cells, in agreement with the view that 3D-primary culture better represents characteristics of tumors in vivo. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely hypoxia, dormancy, anti-apoptotic features and their resulting drug resistance.

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.

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.

BACKGROUND AND OBJECTIVES 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. METHODS We performed PD-1 immunohistochemistry in two cohorts: 40 metastasis-negative SLNs including 10 patients for each subtype (luminal A-like, luminal B-like, HER2, and triple negative breast cancer [TNBC]); and 25 pairs of metastasis-positive SLNs and non-SLNs (10 luminal A-like, 10 luminal B-like, and 5 TNBC). RESULTS Among 40 metastasis-negative SLNs, 34 and 6 samples were PD-1 intensity grade 1 (low) and 2 (high), respectively. PD-1 intensity correlated with PD-1-positive lymphocyte numbers (P = 0.005); TNBC had the highest PD-1 lymphocyte numbers among all subtypes. The median PD-1-positive lymphocyte number was higher in SLNs than non-SLNs. In most cases, more lymphocytes in SLNs expressed PD-1 than those in non-SLNs (P

Introduction: Tumor microenvironment plays a key role in the development and progression of tumors. Although adipose tissue is a predominant component of tumor microenvironment in mammary tissue, and secretes various cytokines, chemokines and growth factors, roles of adipocytes in the breast cancers remain to be elucidated. Methods: In this study, we identified adipsin, an adipokine predominantly expressed in the adipose-derived stem cells (ADSCs) of breast cancer patients, enhanced the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer patient-derived xenograft (PDX) cells. Human breast cancer PDXs were established by the engraftment of surgical specimens of the breast cancer patients into immunodeficient mice. Human mammary adipose-derived stem cells (ADSCs) were isolated from the enzymatically dissociated adipose tissues of the surgical specimens of breast cancer patients. Results: When ADSCs with diverse patient backgrounds were co-cultured with human breast cancer PDX cells, ADSCs significantly enhanced the clonogenicity of breast cancer PDX cells (n = 9; p < 0.05). We found that adipsin, an adipokine that is involved in the generation of C3a and C3b in the complement alternative pathway, was highly expressed in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p < 0.05). Accordingly, the amount of C3a was significantly higher in the cultured supernatants of ADSCs when compared to those of breast cancer PDX cells (n = 9; p < 0.05). Suppression of adipsin by a specific inhibitor or knockdown in mammary ADSCs significantly suppressed the sphere-forming abilities of breast cancer PDX cells; reduced the mRNA expression levels of the cancer stem cell marker CD44 and the chemokine receptor CXCR4 (p < 0.01); and reduced the cell surface expression of CD44 in breast cancer PDX cells, suggesting that cancer stem cell-like properties were regulated by adipsin. Although the suppression of CXCR4 by a specific inhibitor showed a tendency to suppress the sphere-forming abilities of breast cancer PDX cells, the dual suppression of adipsin/C3a and CXCR4 by their specific inhibitors showed no additive effects on the suppression of the clonogenicity of breast cancer PDX cells. Conclusion: These observations suggest that ADSCs are important components of tumor microenvironment in human breast cancers, and secrete adipsin that functions as regulators of the clonogenicity, proliferation, and cancer stem cell-like properties of human breast cancer cells. Citation Format: Hideaki Goto, Yohei Shimono, Toru Mukohara, Yohei Funakoshi, Yoshinori Imamura, Seishi Kono, Shintaro Takao, Akira Suzuki, Hironobu Minami. Adipsin enhanced clonogenicity and proliferation of human breast cancer patient-derived xenograft cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1006.

Natural killer (NK) cells, a component of the innate immunity, play important roles in tumor suppression. In this study, three human breast cancer patient-derived tumor xenografts (PDXs), established by the transplantation of surgical specimens, were passaged in immunodeficient NOD/SCID mice or NSG mice, that further lacks NK cell activity. The intensity of the relative growth suppression between NOD/SCID and NSG mice was clearly different depending on the PDX lines, and it was associated with the intensities of the CD49b-positive NK cell infiltration in the PDX tumor tissues. However, no obvious association was observed between the mRNA expression levels of the NK cell ligands in the PDX tumor cells and the intensity of NK cell infiltration into the PDX tumors. These results suggest that the suppressive effect of NK cells on the growth of breast cancer PDX is highly variable depending on the PDX lines. Further studies are needed to elucidate the molecular mechanism of NK cell infiltration in PDX tumors.

Endocrine treatment-related adverse events have a strong impact on patients’ quality of life and sometimes result in treatment discontinuation. Since joint symptoms are the most frequently recognized side effect of aromatase inhibitors, evaluation of associated risk factors may yield significant findings. A total of 391 postmenopausal Japanese women with estrogen receptor-positive breast cancer and treated with adjuvant anastrozole were enrolled from 28 centers for assessment of patient-reported outcomes (PROs) in this prospective cohort study (SAVS-JP, UMIN000002455). Patients completed the self-report questionnaire at baseline and after 3, 6, 9, and 12 months of treatment for evaluation of frequency of treatment-related joint symptoms (arthralgia, decrease in range of joint motion, and joint stiffness). We obtained PROs from 362 patients (92.6 %) at baseline and at one or more subsequent points. New or worsening from baseline of joint symptoms were reported by 260 patients (71.8 %). More than 90 % of the symptoms were mild or moderate and nearly 80 % had occurred by 6 months. Multivariate analysis showed that a short time span after menopause [odds ratio (OR) 0.95, 95 % confidence interval (CI) 0.90–0.99; P = 0.02] and adjuvant chemotherapy (OR 2.29, 95 % CI 1.06–4.95; P = 0.03) were significant independent risk factors for joint symptoms. No significant relationships between body mass index (BMI) and joint symptoms were identified. Eighteen patients discontinued treatment during the 1st year and eight of them reported joint symptoms. Taking into consideration that PROs may yield higher prevalence rates than physician ratings for symptoms published in pivotal clinical trials, we found that a short time span after menopause and use of adjuvant chemotherapy, but not high BMI, were significantly associated with joint symptoms. These findings might prove useful for counseling before initiating treatment with adjuvant aromatase inhibitors in postmenopausal Japanese women.

Gingival fibromatosis is a rare disease characterized by enlargement of the gingiva. The purpose of this study was to analyze a case of idiopathic gingival fibromatosis, using histochemical and immunohistochemical staining and transmission electron microscopy. The patient was a 39-year-old Japanese man, in whom the gingiva was enlarged throughout the entire mandible and maxilla. Specimens of gingival fibromatosis exhibited epithelial hyperplasia and increased amounts of collagen fiber bundles in the connective tissue light-microscopically. Well-developed collagen bundles were strongly stained with Azan and Masson trichrome staining. Immunohistochemically, the gingival connective tissue was specifically stained by type I collagen and vimentin antibodies. Ultrastructurally, the lesion consisted of fibroblasts and mature collagen fibers running in all directions. No myofibroblasts were detected histochemically, immunohistochemically, or ultrastructurally. These findings suggested that this disease may be the result of an increase in collagen synthesis by the fibroblasts and/or that it may be associated with one of the findings of histologic heterogeneity.

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 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.

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