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Purpose: Elucidating the unique immunomodulatory mechanisms in breast cancer microenvironment should provide useful insights to aid the development of new therapeutic strategies for this disease. Some studies suggested the immune regulatory function of hormone receptor such as estrogen receptor-α (ER) and androgen receptor (AR), but their mechanism has not been fully understood because of the complexity of immune milieu in breast cancer microenvironment. In this study, we systematically analyzed the relationships between ER, progesterone receptor (PgR), and AR expression and the immunological profile in breast cancer tissue. Methods: Gene set enrichment analysis (GSEA) was used to screen the biological processes associated with the expression of human sex hormone receptor genes (ESR1, PGR, and AR), using a gene expression profile dataset of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Then, using METABRIC and a gene expression profile dataset of The Sweden Cancerome Analysis Network - Breast (SCAN-B), the correlation between the immune cell composition in breast cancer tissue (estimated with the CIBERSORTx) and hormone receptor expression was analyzed. In our previous study of 45 breast cancer tissues, we evaluated the level of human tumor infiltrating lymphocytes (hTILs), expression of human programmed death-ligand 1 (hPD-L1), and infiltration of 11 types of immune cells, using hematoxylin–eosin staining, immunohistochemistry (IHC), and multicolor flow cytometry, respectively. In this study, the levels of ER, PgR, and AR expression were further evaluated using IHC, and their relationship with the immunological profile of breast cancer tissues was analyzed. Results: GSEA showed that the expression levels of the ESR1, PGR, and AR genes were negatively correlated with multiple immunological processes, including “INFLAMMATORY RESPONSE.” Analysis of the correlations between the immune cell composition and hormone receptor gene expression showed that ESR1 expression was inversely correlated with Macrophage M1, CD4 memory activated T cells, Macrophage M0, CD8 T cells, and CD4 memory resting T cells; PGR expression was inversely correlated with Macrophage M1, CD4 memory activated T cells, and Macrophage M0; and AR expression was inversely correlated with Macrophage M0 and Macrophage M1. Immunohistochemical evaluation of ER and AR expression revealed both receptors to be inversely associated with hTIL, hPD-L1 expression, and leukocyte infiltration in breast cancer tissue. Analysis of the immune cell composition in these tissues revealed that ER expression was associated with the decreased infiltration of total T cells, CD4+ T cells, monocytes/macrophages, myeloid-derived suppressor cells, dendritic cells, and myeloid dendritic cells; PgR expression was associated with the decreased infiltration of dendritic cells; and AR expression was associated with the decreased infiltration of CD4+ T cells, monocytes/macrophages, nonclassical monocytes, myeloid-derived suppressor cells, dendritic cells, myeloid dendritic cells, and minor natural killer cells. Conclusion: The correlation of hormone receptor expression with specific immunological profiles in the breast cancer microenvironment both at the genetic and protein levels strongly suggests that hormonal signals may preferentially affect certain subsets of immune cells. Citation Format: Toru Hanamura, Shigehisa Kitano, Hiroshi Kagamu, Makiko Yamashita, Mayako Terao, Takuho Okamura, Nobue Kumaki, Katsuto Hozumi, Takayuki Iwamoto, Chikako Honda, Sasagu Kurozumi, Naoki Niikura. Hormone receptor expression is associated with specific immunological profiles in the breast cancer microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-16.

Background: Trastuzumab Deruxtecan (T-DXd) is a novel antibody-drug conjugate that targets human epidermal growth factor receptor 2 (HER2) with a DNA topoisomerase I inhibitor payload and has demonstrated antitumor activity in clinical trials. Based on the results of a global Phase II clinical trial (DESTINY-Breast01, NCT03248492), T-DXd was conditionally approved for the treatment of patients with HER2+ unresectable or metastatic breast cancer who had received ≥2 prior anti-HER2 based therapies (US, EU, UK, etc) or after prior chemotherapy (Japan). With regards to the safety profile, the incidence of cumulative toxicities such as peripheral neuropathy and edema was lower with T-DXd as compared to what has been observed with taxanes. However, chemotherapy-induced nausea and vomiting (CINV), fatigue, and neutrophil count decrease were observed as treatment emergent adverse events (TEAE). Interstitial lung disease (ILD)/pneumonitis was especially observed in 15.2% of patients treated with T-DXd, leading to death in 2.7% of patients. These adverse events need appropriate monitoring and management in daily clinical practice. We hypothesized that electronic patient-reported outcomes (ePROs) monitoring would allow early detection of adverse events due to T-DXd and thus improve patient safety and quality of life (QoL). Study design: This is an open-label, multicenter, randomized controlled study comparing ePRO monitoring with routine follow-up of patients with HER2+ advanced or metastatic breast cancer who receive T-DXd in routine clinical practice. Patients randomized to the ePRO group are monitored through weekly PRO-CTCAE assessment as well as daily temperature and SpO2 measurement using the ePRO application on the patient's mobile device. Changes in symptoms are monitored by the attending physician using an electronic chart. In addition, an e-mail alert is sent to the site medical staff if any symptoms exceed a predetermined threshold. The primary endpoint of the study is the change from baseline in the EORTC QLQ-C30 (Global QoL) score at 24 weeks. Secondary endpoints include time to deterioration of Global QoL, EORTC FA12 score change, overall survival, progression-free survival, and adherence to the ePRO monitoring as assessed by the proportion of patients completing the questionnaires. Analysis of primary endpoint will be done using mixed effects models for repeated measures (MMRM) and sample size will be estimated based on the Wald test for differences between groups in mean parameters at 24 weeks. To detect 10-points score difference between the groups with a standard deviation of 24, with a two-sided alpha level of 10%, and a power of 87%, 55 patients would be required in each group. Enrollment started in March 2021. Clinical trial identification: jRCTs031200387 Citation Format: Takafumi Sangai, Yuichiro Kikawa, Mitsuchika Hosoda, Yohei Hamanaka, Yuko Tanabe, Tatsuya Yoshida, Kaori Tane, Daisuke Takabatake, Tetsuhiko Taira, Kazuhiro Araki, Takayuki Iwamoto, Mamoru Takada, Kazutaka Narui, Takeshi Yamaguchi, Akimitsu Yamada, Tomoko Miura, Yukari Uemura, Tomohiko Aihara, Hirofumi Mukai, Naruto Taira. Randomized study comparing electronic patient reported outcomes (ePROs) monitoring with routine follow up during trastuzumab deruxtecan treatment in patients with inoperable or metastatic breast cancer (PRO-DUCE study) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-12-08.

Background Chemotherapy-induced amenorrhea (CIA) is one of the critical side effects from the chemotherapy in premenopausal patients with breast cancer. The goals of our study are the following: (1) to investigate the factors affecting the incidence of CIA; and (2) to evaluate the prognostic role of CIA in premenopausal patients with breast cancer. Methods We conducted a post hoc retrospective substudy to examine the incidence of the CIA and the relationship between CIA and prognosis in NSAS-BC02 that compared taxane alone to Doxorubicin(A) Cyclophosphamide(C) followed by taxane in postoperative patients with node-positive breast cancer Results Of 395 premenopausal women, 287 (72.7%) had CIA due to protocol treatment. Regarding type of protocol regimen, proportion of CIA was 76.9% in AC Paclitaxel(P), 75.2% in AC Docetaxel(D), 62.8% in PTX, and 75.2% in DTX. Predictive factors of CIA were age increase by 5 years (OR 1.50), ER positivity (OR 2.08), and HER2 3 + ( OR 0.40) according to logistic regression analysis. According to the log rank test and the Cox proportional hazards model, CIA group had significantly better disease-free survival than non-CIA group (P Conclusion Treatment with taxane alone caused high frequency of CIA in premenopausal women with breast cancer. CIA did not turn out to be an independent prognostic factor, taking guarantee-time bias into consideration. Further clinical studies are needed to validate these findings.

Introduction Patients with residual disease usually have a poor prognosis after neoadjuvant chemotherapy for breast cancer. The aim of this study was to explore therapeutic targets and potential additional adjuvant treatments for patients with residual disease after standard neoadjuvant chemotherapy. Patients and Methods We retrieved publicly available complementary DNA microarray data from 399 human epidermal growth factor receptor 2 (HER2)-negative primary breast cancer samples from patients who underwent standard neoadjuvant chemotherapy. We analyzed the messenger RNA (mRNA) expression levels of key breast cancer markers and therapeutic target genes according to residual cancer burden (RCB) classification: RCB-0/I, RCB-II, and RCB-III. Results Among hormone receptor–positive samples, there were more luminal A tumors by PAM50 (Prediction Analysis of Microarray 50 [Prosigna], aka Prosigna Breast Cancer Prognostic Gene Signature Assay) in RCB-III than in RCB-0/I and RCB-II (P < .01). The mRNA expressions of ESR1 and PGR were significantly higher, and that of MKI67 was lower in RCB-II and RCB-III than in RCB-0/I. The mRNA expression of cyclin D1 was up-regulated in RCB-III and that of CDKN2A was down-regulated in RCB-III (P = .027 and < .01). Among triple-negative (TN) samples, RCB-III had higher clinical stage and more lymph node–positive samples than RCB-0/1 and RCB-II (P < .01). In both subtypes, VEGF-C expression was significantly higher in RCB-III than in RCB-0/I and RCB-II. Conclusion In hormone receptor–positive breast cancer, biological features such as luminal A were associated with RCB; this trend was not observed in TN breast cancer. Further, some targeted therapies should be tested as new strategies after standard neoadjuvant chemotherapy in future clinical trials.

Background In hormone receptor (HR)-positive / human epidermal growth factor receptor 2 (HER2)-negative breast cancers, high Ki-67 predicts poor prognosis. Recently, it has been reported that among breast cancers with high Ki-67, low Ki-67 after short-term preoperative hormone therapy (post Ki-67) might predict favorable prognosis compared to high post Ki-67 (SABCS 2017). However, the differences in gene expression profiling of breast cancers between high and low post Ki-67 among high Ki-67 before treatment are unclear. Therefore, this study aimed to clarify genetic differences in two groups and to explore novel therapeutic targets for the poor prognostic group after the treatment. Methods Seventy-seven patients with primary HR-positive / HER2-negative breast cancer who received an aromatase inhibitor for 2 weeks [NCBI Gene Expression Omnibus repository GSE80077:19 cases, GSE20181:58 cases] were enrolled in this study. Forty-five patients with high pre Ki-67 among them were stratified into two groups, high (H→H) and low (H→L) post Ki-67 after short-term preoperative hormone therapy. We compared gene expression profiling in two groups about the followings. 1) 3 genes (ESR1, PGR and ERBB2) related to classical clinical treatment strategy of breast cancer, immune- and inflammatory-related genes 2) 178 pathways (gene set analysis) 3) 41 genes that are targeted by FDA-approved drugs or have been investigated with clinical trials as molecular target agents for various malignant tumors including breast cancer Results 1) TNF that is inflammatory-related gene were significantly overexpressed in H→L group (P=0.021). However, 3 genes related to clinical treatment of breast cancer and immune-related genes expression had no significant difference between two groups. 2) 5 gene sets (Tryptophan metabolism, Propanoate metabolism, beta-Alanine metabolism, SNARE interactions in vesicular transport and Nucleotide excision repair) were significantly upregulated in H→L group. (P ≤ 0.005) 3) 5 targeted genes (PARP, BRCA2, FLT4, CDK6 and PDCD1LG2) were significantly overexpressed in H→H group (P ≤ 0.05). Conclusions There were different gene expression and biological processes in two groups stratified by post-Ki67. In the future, it is necessary to seek new therapeutic strategies for poor prognostic group with high post-Ki67 in considerations of these differences. Citation Format: Yukiko Kajiwara, Takayuki Iwamoto, Yusuke Otani, Miwa Fujihara, Yoko Suzuki, Minami Hatono, Takahiro Tsukioki, Kengo Kawada, Mariko Kochi, Hirokuni Ikeda, Tadahiko Shien, Naruto Taira, Hiroyoshi Doihara. Gene expression profiling of breast cancers between high and low Ki-67 after short-term preoperative hormone therapy among hormone receptor-positive / human epidermal growth factor receptor 2-negative breast cancers with high Ki-67 [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 P5-13-04.

Background: Patient reported outcomes (PROs) have been increasingly important in breast cancer treatment. The questionnaires to evaluate PROs require high response compliance, simplicity, and friendliness. Recently, the communication with Emojis has become friendliness because of the development of social networking services and electronic devices. Therefore, we developed the Emoji scale, a novel indicator to evaluate PROs. Furthermore, two-step validation pilot studies were planned to examine the reliability of the Emoji scale for breast cancer patients at a single center. Pilot 1(Step 1) Aims: To evaluate the content validity (the primary endpoint), concurrent validity and test retest reliability of the Emoji scales (secondary endpoints). Eligibility criteria: (1) Patients with stage 0-III breast cancer. (2) Patients who have been over 1 year since the end of the initial treatment such as surgery, chemotherapy, and radiation therapy or (3) hormone receptor positive patients who have been over 1 year from the start of the endocrine therapy. (4) Patients aged >20 years who understand Japanese. Methods: The participants will complete the PRO Common Terminology Criteria for Adverse Events (PRO-CTCAE) and Emoji scale questionnaires regarding 18 cancer related symptoms twice. First, both questionnaires will be completed in a consulting room while the staff will track the time required for completion of each questionnaire. Thereafter, the patients will be asked if there are any difficult or confused items. Second, both questionnaires will be completed again at home 7 days later. The number of planned participants is 100. Statistical analyses: Content validity will be evaluated by categorizing the comments from participants, and the nature of problems will be determined. The correlation coefficient between the PRO-CTCAE and Emoji scale scores will evaluate the concurrent validity. For the test-retest reliability, the kappa statistic between each scale at different time will be used. We will compare the time required for completion with using the t-test. Pilot 2(Step2) Aims: To evaluate the responsiveness (the primary endpoint), the effect size, the concurrent validity and comparison of the average score between participants with high and low Performance Status. (the secondary endpoints). Eligibility criteria: (1) Patients with stage 0-IV breast cancer who are scheduled to start chemotherapy or endocrine therapy. (2) Patients aged >20 years who understand Japanese. Methods: The participants will complete both the PRO-CTCAE and Emoji scales before they start a new breast cancer treatment (T1). Those will comprise chemotherapy (Cohort A: anthracycline based regimens, Cohort B: docetaxel based regimens, Cohort C: paclitaxel based regimens, Cohort E: eribulin based regimens, and Cohort F: capecitabine/S1 based regimens) or endocrine therapy (Cohort D: tamoxifen/aromatase inhibitor). After several weeks, the participants will complete the same questionnaires again (T2). This time interval will depend on the therapy, and some participants will be complete once again the questionnaires (T3) a few weeks after T2. The number of planned participants is 30 in each cohort. Statistical analyses: Responsiveness will be evaluated by the correlation coefficient between the change from the baseline to second or third score of each scale. The effect size of the Emoji scale will be determined by the difference between the average score of the second or third and that of the baseline divided by the standard deviation of the baseline score. The correlation coefficient for each scale at each time will evaluate the concurrent validity at each time. A comparison of the average scores between PS will be performed using the t-test. Citation Format: Yoko Suzuki, Naruto Taira, Yukiko Kajiwara, Mariko Kochi, Takayuki Iwamoto, Hirokuni Ikeda, Tadahiko Shien, Hiroyoshi Doihara, Yuichiro Kikawa, Yukari Uemura. Validation study of the Emoji scale in evaluating patient reported outcomes for breast cancer patients [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 OT3-19-01.

Purpose The HORSE-BC study previously demonstrated that 2nd-line endocrine therapy (ET) for metastatic breast cancer (MBC) patients with acquired endocrine resistance still provided a clinically meaningful benefit. Herein, we investigated health-related quality of life (HR-QOL) in HORSE-BC. Methods MBC patients with acquired endocrine resistance who were scheduled for 2nd-line ET were recruited. HR-QOL was assessed at baseline and 1 and 3 months after initiation of 2nd-line ET. To investigate the minimally important difference (MID) in the Functional Assessment of Cancer Therapy-Endocrine Symptoms (FACT-ES), we evaluated the means and standard deviations (SDs) for the distribution-based method, and differences in the change in HR-QOL for the anchor-based method. We also investigated the association between FACT-ES total scores and clinical benefit. Results Overall, 56 patients were enrolled. Of these, 47 were analyzed. When defined as 1/3 SD estimates based on the distribution method, the calculated MID was 5.9. The MIDs of the FACT-ES total scores based on the anchor method were 7.7 for decline and 4.1 for improvement. The proportions of MID decline were 6.1% and 14.7% lower in patients who experienced clinical benefits than in those who did not at 1 month and 3 months, respectively. The respective ratios of MID improvement in patients who experienced clinical benefits were 18.3% and 3.2% higher, respectively; mean change in FACT-ES total score from baseline was improved in patients who experienced clinical benefits. Conclusion Maintaining HR-QOL as determined by FACT-ES may be associated with clinical benefits in patients with acquired endocrine-resistant MBC treated with ET.

Adding taxane to an anthracycline-based regimen improves survival in node-positive breast cancer patients, as shown by clinical trials and meta-analyses. However, no studies have analyzed the number of metastatic lymph nodes in patients with estrogen receptor (ER)-positive cancer. This study investigated whether adding a taxane to an anthracycline-based regimen improved prognosis in node-positive, ER-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer patients in a real-world setting. Using Japanese Breast Cancer Society registry data, we compared disease-free survival (DFS) of patients with ER-positive, HER2-negative breast cancer, excluding those receiving neoadjuvant chemotherapy, between those who received an anthracycline-based regimen followed by a taxane-based regimen (A + T) and those who received only an anthracycline-based regimen (A w/o T), stratified by lymph node status. A Cox proportional hazards model was used to evaluate DFS in both groups. There were 4566 eligible patients with ER-positive, HER2-negative breast cancer. During the median follow-up period of 60 months, there were 481 recurrences and 149 deaths. There was no significant difference in DFS between the A + T and A w/o T groups among patients with 1–3 positive nodes, while there was a significant difference among patients with ≥ 4 positive nodes. In patients with ER-positive, HER2-negative breast cancer, adding taxane to an anthracycline regimen did not improve DFS in patients with metastasis in 1–3 lymph nodes. We considered that the group without the addition of taxane might be present in patients with ER-positive, HER2-negative lymph node metastases.

Background: Lymphocyte predominant breast cancer subgroup, defined as ≥ 50% stromal tumor-infiltrating lymphocytes (sTILs), is associated with high pathological complete response (pCR) rate after neoadjuvant therapy (NAT) and favorable outcome. In a cohort of triple negative (TNBC) and HER2+ breast cancer (BC) patients treated with NAT, we aimed to assess the predictive and prognostic value of pre- and post-NAT sTILs and the information provided by the change in sTILs during NAT. Materials and methods: Two-hundred and nine consecutive patients (n=80 TNBC; and n=129 HER2+) who received NAT between 2001 and 2009 in our institution were evaluated. Pre-NAT sTILs were assessed on biopsy sample (baseline) and post-NAT sTILs on surgical specimens just for non-pCR patients. sTILs level was categorized as low 0-9%, intermediate 10-49%, and high ≥50%. The change in sTILs during NAT was calculated as the absolute difference between pre- and post-NAT sTILs. We evaluated the association of pre-NAT sTILs and pCR, and the association between pre- and post-NAT sTILs, and their change with relapse-free survival (RFS). Results: Overall pCR rate was 37.8% (31.3% for TNBC, 41.2% for ER+/HER2+BC, 42.3% for ER-/HER2+BC). In each subtype, pre-NAT low sTILs group was significantly associated with lower pCR rate. During the median follow-up period of 98 months, 44 recurrences (21.1%) were observed. For TNBC, low pre-NAT sTILs group was associated with higher recurrence risk compared with int/high sTILs (HR=4.675 [2.013-10.859], p Conclusion: In TN and HER2+ BCs, tumors with low pre-NAT sTILs have a low likelihood to achieve a pCR (predictive marker). In TNBC, low pre-NAT sTILs were associated with higher recurrence risk. In non-pCR TNBC patients, both low pre- and post-NAT sTILs were associated with shorter RFS. These results suggest that sTILs information should be taken into account when additional post-surgery treatments are considered in non-pCR patients. Citation Format: Ochi T, Giampaolo B, Murai M, Nozaki F, Kobayashi D, Iwamoto T, Niikura N, Suzuki K, Yamauchi H, Hayashi N. Predictive and prognostic value of stromal tumor-infiltrating lymphocytes before and after neoadjuvant therapy in triple negative and HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-31.

Background: Overall survival (OS) is the established endpoint to evaluate the effects of drug treatment in comparative clinical trials of metastatic breast cancer. But assessing OS requires long follow-up periods and large sample size, which raise costs and create long delays in the drug approval process. Progression-free survival (PFS) or time to progression (TTP) is considered as a surrogate for OS and is often used as an alternative to OS. In some cancers the two endpoints are highly correlated, but in others they are not. Furthermore, the effect of breast cancer (BC) subtypes on the surrogacy of PFS/TTP for OS has not been completely defined. Method: A systematic literature review of randomized control trials was conducted to identify studies that reported both the hazard ratio (HR) of PFS/TTP and OS for BC subtypes {i.e. estrogen receptor (ER) positive, HER2 positive, and triple negative (TN)}. The correlation between the HR of PFS/TTP and OS was evaluated using weighted Spearman's rank correlation. Results: A total of 49 trials (34 phase III trials and 15 phase II trials) were selected for analysis. Among these trials, there were 8 comparison trials between one chemotherapy and another chemotherapy regimen, 18 comparison trials between chemotherapy and chemotherapy plus molecularly-targeted therapy, 9 comparison trials between one endocrine therapy and another endocrine therapy, and 5 comparison trials between endocrine therapy and endocrine therapy plus molecularly-targeted therapy. There were 17 trials reporting the HR of PFS/TTP and OS for ER positive, 16 trials for HER2 positive, and 9 trials for TN BC. Weighted Spearman's rank correlation revealed that coefficient between the HR of PFS/TTP and OS was 0.721(p Conclusion: There was a strong correlation between the HR of PFS/TTP and OS for ER positive BC, and a weak correlation between the HR of PFS/TTP and OS for HER2 positive and TN BC. The validity of using PFS/TTP as an OS surrogate marker was shown for metastatic BC, especially for ER positive BC. Citation Format: Tsukioki T, Taira N, Sakamaki K, Suzuki Y, Kajiwara Y, Hatono M, Takahashi Y, Kawata K, Kochi M, Iwamoto T, Ikeda H, Shien T, Doihara H. Progression-free survival or time to progression in comparative clinical trials of metastatic breast cancer as a potential surrogate for overall survival: A systematic review of 49 trials focusing on breast cancer subtype [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-19.

Background : Isoflavones are phytoestrogens that may be effective in preventing osteoporosis, reducing cardiovascular events, and improving menopausal symptoms such as hot flash due to their estrogen-like actions. Isoflavones are currently used as a supplement for improving symptoms of menopause. Epidemiological studies have shown that ingestion of soy products may also reduce the risk of breast cancer, and antitumor effects on hormone receptor (HR)-positive breast cancer cells have been shown in vitro for equol, an isoflavone metabolite. Equol binds to estrogen receptors (ERα, β) and is thought to antagonize ERα-estradiol binding in the presence of estradiol. Since equol shows an antiestrogenic effect, similar to that of the hormonal agent tamoxifen, it is thought that equol acts as a selective estrogen receptor modulator (SERM), but the mechanism is still unclear. In this study, we evaluated the antitumor effects of equol alone and in combination with existing therapeutic agents in HR-positive breast cancer cells, and examined the mechanism of these effects. Methods : The antitumor effects of equol alone and in combination with hormone drugs (4-hydroxytamoxifen (Tam), fulvestrant (Ful)) and chemotherapeutic agents (paclitaxel (Ptx), doxorubicin (Dox)) were examined using a MTS assay. Combination indexes (CIs) were determined in HR-positive MCF-7, T-47D, and ZR-75-1 cell lines. The mechanisms of the drug effects were evaluated by Western blot for assessment of changes in chemoresistance factors at the protein level. Results : In MCF-7, T-47D and ZR-75-1 cells, there was a concentration-dependent antitumor effect of equol and of the other hormonal agents. CIs showed an antagonistic effect of equol with Tam and a synergistic effect with Ful in all cell lines. Equol also had an antagonistic effect with the two chemotherapeutic agents, with the strongest antagonism occurring at a low dose of equol. Western blot showed that ER, PgR, Cyclin D1 and Bcl-2 were upregulated via ER at a low concentration of equol, similarly to the effect of 17-β-estradiol (E2), and ER, PgR, Cyclin D1 and Bcl-2 were downregulated at a high concentration, similarly to the effect of Tam. In addition, ER, PgR and cyclin D1 were downregulated with the combination of equol and Tam, while the expression of Bcl-2, a chemoresistance factor, increased. Conclusion : These results suggest that equol has a concentration-dependent antitumor effect in HR-positive breast cancer cell lines and may antagonize the effect of existing therapeutic agents (hormone drugs and chemotherapeutic agents). In particular, it was considered that in combination of equol and Tam, the apoptosis inducing action of each drug was attenuated and antagonistic effect was shown. Citation Format: Hatono M, Ikeda H, Taira N, Suzuki Y, Kajiwara Y, Kawata K, Takahashi Y, Tsukioki T, Kochi M, Iwamoto T, Shien T, Doihara H. Effects of a soy isoflavone in breast cancer treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-05-06.

Background:It is unclear how to define responsiveness to endocrine therapy (ET) during the clinical course of advanced breast cancer (ABC), especially in evaluation of the effect of sequential ET. Objective:The goal of the study was to evaluate the efficacy of second line treatment of physician's choice (2nd-line TPC) for estrogen receptor-positive (ER+) and HER2-negative postmenopausal ABC with very low or low sensitivity to initial ET. Methods:A multicenter prospective observational cohort study was performed for 2nd-line TPCs. ABC with low sensitivity to initial ET was defined as recurrence within 5 years (yrs) during adjuvant ET or progression within 9 months (mo.) of initial ET. Similarly, ABC with very low sensitivity to initial ET was defined as recurrence within 2 yrs during adjuvant ET or progression within 3 mo. of initial ET. The expected clinical benefit rate (CBR: defined as patients who achieved CR, PR or SD for 24 weeks) was 50%. The null hypothesis of a CBR of 30% was tested with a one-sided α of 5%. 90% confidence intervals (CIs) were calculated for hypothesis tests. Results: A total of 56 patients (pts) were enrolled, but 7 were ineligible and one discontinued before starting the protocol treatment. The median age was 66 yrs (range: 41-88) and the median BMI was 23.4 kg/m2 (16.4-31.9). All pts were ER+ and 80% were PgR+. Most of pts had a baseline PS of 0 or 1, 90% had invasive ductal carcinoma, and 10% had invasive lobular carcinoma. Postoperative recurrence was detected in 84% and these pts had a median duration of adjuvant ET of 30.5 mo. (5.3-58.9). De novo stage IV ABC was present in 16%, with a median duration of first-line ET of 5 mo. (2.3-10.8). Adjuvant chemotherapy including anthracycline- and/or a taxane-containing regimen was administered in 58% (29/49). As adjuvant ET before initial recurrence, 34 pts received non-steroidal aromatase inhibitors (AIs) (88.0%), 1 received a steroidal AI (2.3%), and 3 received a selective estrogen receptor modulator (SERM). As first line ET in de novo stage IV, 7 pts (14%) were treated with AIs or a SERM (1 case). 2nd-line TPCs were also used, with 40 pts receiving fulvestrant (82%), 5 receiving SERMs (10%), 3 receiving a mTOR inhibitor plus a steroidal AI (6%), and one patient receiving an AI alone. The overall CBR was 44.9% (90% CI: 34.6-57.6, p=0.009), and CBR was similar across following subgroups (PgR+: n=39, 51.3%, 90% CI: 39.6-65.2, p=0.0016; very low sensitivity group: n=17, 58.8%, 90% CI: 42.0-78.8, p=0.003; non-visceral metastases: n=25, 40%, 90% CI; 34.1-65.9, p=0.0175). However, there were not statistically significant CBR in PgR- (n=10, 20.0%, 90% CI; 8.73-50.7, p=0.617), fulvestrant subgroup (n=40, 40.0 %, 90% CI; 29.2-54.2, p=0.063), low sensitive group (n=32, 37.5%, 90% CI; 26.0-53.6, p=0.1326), and visceral metastases (n=24, 48%, 90%CI; 28.2-60.3 p=0.072). The median PFS was 7.1 mo. (95% CI: 5.6-10.6). Conclusion:This study shows that 2nd line ETs was effective and might be a valid option in the sequence of treatments for postmenopausal women with ABC with low sensitivity to initial ET. It was suggested that PgR and visceral metastasis were significant predictive factors for CBR. Citation Format: Araki K, Fujisawa T, Sakamaki K, Kikawa Y, Iwamoto T, Sangai T, Shien T, Takao S, Nishimura R, Takahashi M, Aihara T, Mukai H, Taira N. Sequential second line endocrine therapy is still an effective strategy for postmenopausal ER+ and HER2- advanced breast cancer with low sensitivity to initial endocrine therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-13-09.

Introduction The previous randomized phase 3 trial (SELECT BC) showed that S-1 as a first-line chemotherapy for metastatic breast cancer (MBC) is non-inferior to taxane with respect to overall survival. This study aimed to identify the usefulness of metabolism-related genes as predictive biomarkers for the response to S-1 compared with taxane using tumor tissue samples from the previous trial. Patients and Methods In this SELECT BC-EURECA study, 147 patients with human epidermal growth factor 2 (HER2)-negative MBC who received either S-1 or taxane were evaluated. Formalin-fixed paraffin-embedded specimens were collected, and 14 genes involved in the pyrimidine metabolic pathway, estrogen receptor, progesterone receptor, HER2, Ki67, and beta-tubulin were measured using reverse transcription polymerase chain reaction in microdissected tumor specimens. The expression of each gene was categorized as low, intermediate, and high by tertile values. Results Interaction tests to identify biomarkers for the response to S-1 compared with taxane, revealed the following as the top 3 biomarkers: RRM1 (P value = 0.24), GGH (P value = 0.25), and MTHFR (P value = 0.28). In the S-1 group, lower GGH and higher MTHFR expression were significantly correlated with better time to treatment failure. In the taxane group, there was no gene that was identified as a significant indicator of treatment failure. Conclusion This biomarker analysis from SELECT BC did not identify any predictive biomarkers for the response to S-1 compared with taxane. Future studies with larger sample size and information on not only mRNA, but also protein and DNA for broad functional analyses are needed.

Recent genome-wide association studies have shown that many single-nucleotide polymorphisms (SNPs) are associated with breast cancer risk. However, it is often unclear how these SNPs are related to breast cancer. Analysis of associations between SNPs and phenotypes may be important for determining mechanisms of action, including carcinogenesis. In previous case–control studies, we found three SNPs (rs2046210, rs3757318, and rs3573318) associated with breast cancer risk in Japanese women. Among these SNPs, two (rs2046210 and rs3757318) are located at 6q25.1, in proximity to the estrogen receptor 1 gene (ESR1). Using data from these studies, we examined associations between factors related to breast cancer risk, such as height, weight, and breast density, and the three SNPs in cases and controls. We also investigated whether the SNPs correlated with breast cancer features, such as estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor type-2 (HER2) status, and clinical stage. There was a significant difference in mean height between risk and non-risk allele carriers for rs2046210 (156.0 ± 5.8 vs. 154.3 ± 5.5 cm, p = 0.002), and rs3757318 (155.8 ± 5.7 vs. 154.7 ± 5.6 cm, p = 0.035) in cases, but no significant associations between height and these SNPs in controls. There was also a significant difference in breast density between risk and non-risk allele carriers for rs2046210 (p = 0.040) and rs3757318 (p = 0.044) in cases. rs2046210 and rs3757318 risk allele carriers tended to have higher breast density in all subjects and in controls. In cases, rs3757318 risk allele carriers were also significantly more likely to be ER-negative compared to non-risk allele carriers (ER-positive rate: 77% vs. 84%, p = 0.036). SNPs rs2046210 and rs3757318, which are associated with breast cancer risk in Japanese women, were significantly associated with height and high breast density, and this association was particularly strong in those with breast cancer. These findings suggest that SNPs in the ESR1 gene region affect phenotypes such as height and breast density.

Breast cancer survival outcomes vary across different ethnic groups. We clarified the differences in clinicopathological and survival characteristics of breast cancer among Japanese, US residents with Japanese origin (USJ), and US residents with other origins (USO). Using Surveillance, Epidemiology, and End Results (SEER) 18 dataset and Japanese Breast Cancer Society (JBCS) registry, we included patients first diagnosed with breast cancer between 2004 and 2015. We categorized the patients into three groups based on the database and the recorded ethnicity: Japanese (all those from the JBCS registry), USJ (those from SEER with ethnicity: Japanese), and USO (those from SEER with ethnicity other than Japanese). Excluding patients diagnosed after 2012, stage 0, and 4 patients, we examined the overall survival (OS) and breast cancer-specific survival (BCSS) using the Kaplan–Meier method and Cox proportional hazards models, adjusting for age, sex, cancer stage, and hormone receptor (HR) status. We identified 7362 USJ, 701,751 USO, and 503,013 Japanese breast cancer patients. The proportion of HR-positive breast cancer was the highest among USJ (71%). OS was significantly longer among Japanese and USJ than USO (Hazard ratio 0.46; 95% Confidence Interval [CI] 0.45–0.47 for Japanese and 0.66 [95% CI 0.59–0.74] for USJ) after adjusting for baseline covariates. BCSS was also significantly higher in the two groups (HR 0.53 [95% CI 0.51–0.55] for Japanese and 0.53 [95% CI 0.52–0.74] for USJ). In stage I–III breast cancer, Japanese and US residents with Japanese origin experienced significantly longer survival than US residents with non-Japanese origins.

Purpose Metformin has been suggested to possibly reduce cancer risk. However, the mechanism underlying the positive effects of metformin on cancer treatment remains unclear. We conducted a prospective study to evaluate the effects of preoperative metformin in patients with early breast cancer. Method We evaluated the effects on immunological factors (TILs, CD4 + , CD8 + , PD-L1, IFNγ and IL-2) by comparing core needle biopsies (CNB) obtained before metformin treatment with surgical specimens. Seventeen patients were enrolled in this prospective study from January to December 2016. We also analyzed 59 patients undergoing surgery during the same period to reveal the correlation of immune factors between CNB and surgical specimen. Result There was a moderate correlation between CNB and surgical specimens on TILs and CD8 + lymphocyte. (TILs Rs = 0.63, CD4 + Rs = 0.224, CD8 + Rs = 0.42) In the metformin group, TILs increases were confirmed in five (29%) patients, while a decrease was confirmed in two (12%). The expressions of CD4 + and CD8 + by TILs were increased in 41% and 18% of surgical specimens, respectively. However, TILs number (p = 0.0554), CD4+ (p = 0.0613) and CD8 + (p = 0.0646) expressions did not significantly increased. Furthermore, IFNγ expression appeared to be increased in response to metformin (p = 0.08). Conclusion Preoperative metformin tends to increase TILs, as well as the numbers of CD4 and CD8 positive lymphocytes, and IFNγ levels. Metformin might improve immune function and have a possibility of chemo-sensitivity and thereby increase the effectiveness of immunotherapy, based on the results of this preliminary study.

Purpose Second-line endocrine therapy (ET) for estrogen receptor (ER)-positive and human epidermal growth factor 2 (HER2)-negative metastatic breast cancer (MBC) is offered based on the response to first-line ET. However, no clinical trials have evaluated the efficacy and safety of secondary ETs in patients with poor responses to initial ET. This study evaluated the efficacy of second-line ET in ER-positive and HER2-negative postmenopausal MBC patients with low or very low sensitivity to initial ET. Methods This multicenter prospective observational cohort study evaluated the response of 49 patients to second-line ETs in postmenopausal MBC patients with low or very low sensitivity to initial ET. The primary endpoint was the clinical benefit rate (CBR) for 24 weeks. Results Of the 49 patients assessed, 40 (82%) received fulvestrant in the second line, 5 (10%) received selective estrogen receptor modulators, 3 (6%) received aromatase inhibitors (AIs) alone, and 1 received everolimus with a steroidal AI. The overall CBR was 44.9% [90% confidence interval (CI): 34.6–57.6, p = 0.009]; CBR demonstrated similar significance across the progesterone receptor-positive (n = 39, 51.3%, 90% CI: 39.6–65.2, p = 0.002), very low sensitivity (n = 17, 58.8%, 90% CI: 42.0–78.8, p = 0.003), and non-visceral metastases (n = 25, 48.0%, 90% CI: 34.1–65.9, p = 0.018) groups. The median progression-free survival was 7.1 months (95% CI: 5.6–10.6). Conclusion Second-line ET might be a viable treatment option for postmenopausal patients with MBC with low and very low sensitivity to initial ET. Future studies based on larger and independent cohorts are needed to validate these findings.

Epidemiological studies have suggested that intake of soy isoflavones is associated with a reduced risk of development of breast cancer and an improved prognosis in patients with breast cancer. In addition, basic research has demonstrated the antitumor effects of these compounds on breast cancer cell lines. However, the detailed effects of the intake of equol, which is one of the metabolites of the soy isoflavones, are yet to be clarified on the risk of development and recurrence of breast cancer and its interactions with drugs used for treating breast cancer. This study aimed to determine the antitumor effects of equol and investigate the impact of adding equol to therapeutic agents for breast cancer using breast cancer cell lines. We examined the antitumor effect of equol on breast cancer cell lines using MTS assay. We also studied the combined effect of equol and the existing hormonal or chemotherapeutic agents using combination index. We evaluated the expressions of the related proteins by Western blot analysis and correlated the findings with the antitumor effect. Equol showed bi-phasic protumor and antitumor effects; at a low concentration, it promoted the tumor growth in hormone receptor-positive cell lines, whereas antitumor effects were generally observed when an excessive amount of dose unexpected in the blood and the tissue was administered. When used with tamoxifen, equol might have some antagonistic effect, although it depends on equol concentration and the type of cancer cells. We confirmed that equol has dual action, specifically a tumor growth-promoting effect and an antitumor effect. Although the results suggested that equol might exert an antagonistic effect against tamoxifen depending on the concentration, equol did not exert an antagonistic effect on other therapeutic agents.

Routine analysis of Ki-67 is not widely recommended for clinical decision-making because of poor reproducibility. Furthermore, counting numerous cells can be laborious for pathologists. Digital image analysis for immunohistochemical analysis was recently developed; however, the clinical efficacy of the Ki-67 index obtained using image analysis is unknown. We retrospectively identified female patients with breast cancer with immunohistochemical Ki-67 and survival data using the pathology database at the Tokai University, Japan. Ki-67 expression was scored by three pathologists. Slides were scanned and converted to virtual slides; Ki-67-positive cells were counted using image analysis. Ki-67 indices obtained by the pathologist’s scoring and image analysis were evaluated by 2 × 2 analysis. Relationships between Ki-67 index and survival outcomes were evaluated using the Kaplan–Meier method and compared using the log-rank test. Based on the 2 × 2 analysis, Ki-67 index obtained using image analysis was moderately correlated with the pathologist’s scoring for all patients (κ 0.41; sensitivity, 0.573; specificity, 0.878). Poorer relapse-free survival was associated with high Ki-67 index than with low Ki-67 index for estrogen receptor-positive, human epidermal growth factor receptor 2-negative, and stage I or II patients scored by pathologists (p

Inflammatory breast cancer (IBC) accounts for 2% of breast cancers but 10% of breast cancer-related deaths in the US. Clinical hallmarks of IBC are tumor cell emboli in lymphatic vessels and overexpression of E-cadherin, which promotes cell clustering. Given these hallmarks, IBC is thought to spread via collective invasion and cell clusters. However, we showed that IBC cells underwent epithelial-to-mesenchymal transition (EMT) and metastasized through EMT. Thus, there are two contradictory theories of IBC metastasis. The objectives of this study were 1) to propose a model that reconciles these two models, and 2) to identify target molecules for inhibition of IBC metastasis. Methods: We previously showed that Matrigel culture induced EMT-like changes in SUM149 IBC cells. To test if this transformation from epithelial (E) to mesenchymal (M) in Matrigel culture is unique to IBC cells, a panel of breast cancer cells was cultured in both monolayer and Matrigel-coated plates. The cells were IBC (SUM149, SUM190, KPL4, IBC3), triple-negative breast cancer (TNBC) (MDA-MB-231, MDA-MB-468), and ER+ (MCF7) cells. Phenotypic changes in morphology and expression of EMT markers (E-cadherin, vimentin) were captured with bright field and immunofluorescent (IF) images, respectively. For genome-wide and targeted transcriptional analysis, SUM149 cells cultured in monolayer and Matrigel were processed using DNA microarrays and Taqman qRT-PCR. To correlate the Matrigel gene signature with M features in human breast cancer, a human breast cancer data set was hierarchically clustered with the Matrigel gene signature. Results: SUM149 cells showed a remarkable phenotypic change from E in monolayer culture to M in Matrigel. IF analysis confirmed induction of vimentin expression in Matrigel but stable expression of E-cadherin (thus, we refer to this state as E/M hybrid). This trend was also observed with SUM190 cells. Using qRT-PCR, we confirmed downregulation of E-cadherin and upregulation of M markers (vimentin, Twist1, Snail1, ZEB2) in Matrigel-cultured SUM149 and SUM190 cells compared to monolayer-cultured cells. DNA microarray transcriptional analysis confirmed this trend in SUM149 cells. TNBC has more M-like features than other breast cancer subtypes. Given this evidence, we clustered human breast cancer data using overexpressed genes in Matrigel-cultured SUM149 cells. We identified a cluster of 20 genes in TNBC samples and, assuming that these genes are drivers of E to E/M transition, chose the inflammation-related gene CSF1 as a candidate. The CSF1/CSF1R axis was inhibited by a CSF1R inhibitor, BLZ945; moreover, treatment with BLZ945 reversed the EMT changes in cells in Matrigel culture. Treatment with 5 μM BLZ945 re-induced E-cadherin expression and suppressed Snail1 and Twist1 expression in Matrigel-cultured SUM149 cells. Conclusion: IBC cells are more prone to undergo transition from E to E/M phenotype in Matrigel culture than are cells of other breast cancer subtypes. The CSF1/CSF1R axis plays a role in this E to E/M transition, thus warranting testing its significance using an in vivo IBC model. Phenotypic transition and reversion between E and E/M phenotypes could be a new paradigm that reconciles two contradictory models of IBC metastasis. Citation Format: Kai K, Iwamoto T, Zhang D, Rao AUK, Thompson A, Sen S, Ueno NT. CSF1/CSF1R axis reprograms epithelial-to-mesenchymal phenotypes in inflammatory breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-14-02.

Although 1% has been used as cut-off for estrogen receptor (ER) positivity, several studies have reported that tumors with ER 1% have characteristics similar to those with 1% ≤ ER 10%. We hypothesized that in patients with human epidermal growth factor 2 (HER2)-negative breast cancer, a cut-off of 10% is more useful than one of 1% in discriminating for both a better pathological complete response (pCR) rate to neoadjuvant chemotherapy and a better long-term outcome with adjuvant hormonal therapy. Our objectives were to identify a percentage of ER expression below which pCR was likely and to determine whether this cut-off value can identify patients who would benefit from adjuvant hormonal therapy.Patients with stage II or III HER2-negative primary breast cancer who received neoadjuvant chemotherapy followed by definitive surgery between June 1982 and June 2013 were included. Logistic regression models were used to assess the association between each variable and pCR. Cox models were used to analyze time to recurrence and overall survival. The recursive partitioning and regression trees method was used to calculate the cut-off value of ER expression.A total of 3055 patients were analyzed. Low percentage of ER was significantly associated with high pCR rate (OR = 0.99, 95% CI = 0.986-0.994, P 0.001). The recommended cut-off of ER expression below which pCR was likely was 9.5%. Among patients with ER ≥ 10% tumors, but not those with 1%≤ER 10% tumors, adjuvant hormonal therapy was significantly associated with long time to recurrence (HR = 0.24, 95% CI = 0.16-0.36, P 0.001) and overall survival (HR = 0.32, 95% CI = 0.2-0.5, P 0.001).Stage II or III HER2-negative primary breast cancer with ER 10% behaves clinically like triple-negative breast cancer in terms of pCR and survival outcomes and patients with such tumors may have a limited benefit from adjuvant hormonal therapy. It may be more clinically relevant to define triple-negative breast cancer as HER2-negative breast cancer with 10%, rather than 1%, of ER and/or progesterone receptor expression.

The present study evaluated whether morphological-measured stromal and intra-tumour tumour-infiltrating lymphocytes (TILs) levels were associated with gene expression profiles, and whether TILs-associated genomic signature (GS) could be used to predict clinical outcomes and response to therapies in several breast cancer subtypes. We retrospectively evaluated haematoxylin eosin (HE)-TILs levels and gene expression profiling data from 40 patients with primary breast cancer and extracted the 22 overexpressed genes in cases with high TILs scores as the TILs-GS. The TILs-GS were compared with breast cancer subtype and were evaluated predictive values for prognosis and response to therapies. Higher TILs-GS expressions were observed for triple-negative and human epidermal growth factor receptor 2 (HER2) positive (+) breast cancers, compared to the luminal types (P

[Background] Past studies revealed that a moderate to high level of physical activity after diagnosis of breast cancer reduces both the risk of breast cancer-related death and death from all causes. Furthermore, some randomized studies suggested that exercise programs improve the percentage of patients who complete the chemotherapy and quality of life, and decrease fatigue, and adverse events. The issues to be determined include defining an established uniform exercise program and the efficacy of a long-term exercise program after breast cancer surgery. [Object] To elucidate the efficacy of a long-term exercise program and to verify the safety and feasibility of a uniform exercise program using an ‘existing social resource’ after primary therapy of breast cancer. [Design] A multi-center, randomized trial. [Method] Subjects: The subjects included patients who had completed treatment for primary breast cancer, including surgery and/or adjuvant chemotherapy. Patients with metastatic breast cancer were excluded. Randomization & intervention: The patients were randomly assigned to three groups. The first group followed an exercise program at Curves® that involved 30 minutes of exercise, including aerobics, weight training, and stretching 3 times a week for 4 months. The second group was given life-style guidance at least once that patients participate in a lecture program about recommended exercise at this point and the importance of weight control after diagnosis of breast cancer using a brochure. The third group served as controls that the patients receive a brochure used same one in the second group. The variables included age and weight. Outcome: The primary endpoint is level of physical activity at 1 year after randomization, and the secondary endpoints are the percentage of those completing the exercise program, patient reported outcomes (QOL, cancer or treatment associated symptoms, fatigue, depression, and anxiety), body mass index, bone density, and level of lymphedema. Period of research: The study will last 2 years beginning March 2016. Sample size: We plan to enroll 400 patients to detect 20% difference with 90% power. Additional study: Some biochemical markers in the blood will be evaluated to determine the mechanism of the effect of exercise on the human body. Citation Format: Kawada K, Taira N, Hatono M, Takahashi Y, Miyoshi Y, Nogami T, Iwamoto T, Motoki T, Sien T, Matsuoka J, Doihara H, Ikeda M, Ogasawara Y, Takabatake D, Yoshitomi S, Kiyoto S, Yamamoto S, Mizota Y, Oka K. Influence of exercise or educational programs on long-term physical activity by patients after surgery for primary breast cancer: A randomized trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT3-07-02.

Background: Breast cancer bone metastasis (BCBM)-specific genes have been reported without considering biological differences based on estrogen receptor (ER) status. The aims of this study were to identify BCBM-specific genes using our patient dataset and validate previously reported BCBM-specific genes, and to determine whether ER-status-related biological differences matter in identification of BCBM-specific genes. Methods: We used Affymetrix GeneChips to analyze 365 primary human epidermal growth factor receptor 2 (HER2)-negative invasive breast cancer specimens. Genes that were differentially expressed between patients who developed bone metastasis and those who developed non-bone metastasis were identified using Cox proportional hazards model, and differential expression of gene sets was assessed using gene set analysis. We performed gene set analysis to determine whether biological function associated with bone metastasis were different by ER status using 2,246 functionally annotated gene sets assembled from Gene Ontology data base. Results: Among 16,712 probe sets, 592 were overexpressed in the bone metastasis cohort compared to the non-bone-metastasis cohort (false discovery rate ≤ 0.05). However, no BCBM-specific genes met our significance tests when the cancers were stratified by ER status. In ER-positive and ER-negative breast cancers, 151 and 125 gene sets, respectively, were overexpressed for BCBM and the majority of BCBM-related pathways were different. Of significant gene sets, only 13 gene sets were overlapped between ER-positive and -negative cohorts. Conclusion: ER-positive and ER-negative breast cancers have different biological pathways in BCBM development. We have yet to explore BCBM-related biomarkers and targets considering the biological features associated with BCBM depending on the ER status.

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2+, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8+ and CD4+ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2+ and TN breast cancers have a lower percentage of TILs and CD8+ and CD4+ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.

Mammography is the standard examination for breast cancer screening of woman aged ≥ 40 years. High breast density on mammography indicates that mammary gland parenchyma occupy a high percentage of the breast. The objective of this study was to investigate factors associated with breast density and the risk of high breast density for breast cancer. A multicenter case–control study was performed in 530 patients and 1043 controls. Breast density was classified as C1–C4 using the Breast Imaging Reporting and Data System (BI-RADS). Clinical factors were obtained from questionnaires or medical records, and the influence of each factor (breast density, menopausal status, body mass index (BMI), parity, presence or absence of breastfeeding history, age at menarche, age at first birth, and familial history of breast cancer) on breast cancer risk in all patients was calculated as an age-adjusted odds ratio (OR). Multivariate logistic regression analyses were then performed in all patients and in pre- and postmenopausal and BMI-stratified groups using factors with a significant age-adjusted OR as adjustment factors. Age-adjusted ORs for breast cancer were significant for breast density, BMI, parity, and breast feeding, but not for age at menarche, age at first birth, or family history of breast cancer. In multivariate analysis, there was a significant correlation between breast density and breast cancer in postmenopausal women (OR for C1 vs. C2 1.90 [95% CI 1.34–2.70]; C1 vs. C4 2.85 [95% CI 1.10–7.16]). This correlation was also significant in patients in the third BMI quartile (22.3–24.5 kg/m2) (OR for C1 vs. C4 8.76 [95% CI 2.38–42.47]); and fourth BMI quartile (>24.5 kg/m2) (OR for C1 vs. C2 1.92 [95% CI 1.17–3.15]; C1 vs. C4 11.89 [95% CI 1.56–245.17]). Breast density on mammography is a risk factor for breast cancer after adjustment for other risk factors. This risk is particularly high in postmenopausal women and those with a high BMI.

Aim Lymphocyte predominant breast cancer (BC) is associated with higher pathological complete response (pCR) rate after neoadjuvant therapy (NAT) and favorable outcome in triple negative breast cancer (TNBC) and HER2+ BC. The predictive and prognostic impact of stromal tumour-infiltrating lymphocytes (TILs) after NAT and the change of TILs before (pre-) and after (post-) NAT are not well studied. We aimed to assess the predictive and prognostic value of pre- and post-NAT TILs, as well as their pharmacodynamics modulation and their change for TNBC and HER2+ BC. Materials and methods Two-hundred and nine consecutive patients (n = 80 TNBC, n = 129 HER2+ BC) who received NAT between 2001 and 2009 in a single institution were included. We evaluated the association between pre-NAT TILs and pCR, and the association between pre- and post-NAT TILs, as well as their immunodynamics change with relapse-free survival (RFS) for patients with residual disease (RD). Results Low pre-NAT TILs compared to int/high were significantly associated with lower pCR rate (TNBC: 4.0% vs 43.6%; HER2+ BC: 26.0% vs 51.9%). The median follow-up period was 98 months. In TNBC with RD, low pre-NAT TILs showed significant association with shorter RFS (HR = 3.844 [1.190–12.421], p = 0.024) in multivariate analysis. Low post-NAT TILs showed borderline significant association with shorter RFS (HR = 2.836 [0.951–8.457], p = 0.061). The change in TILs was not associated with RFS. In HER2+ BC, low pre-NAT TILs were not associated with RFS. Conclusion In TN and HER2+ BCs, low pre-NAT TILs tumours had a low likelihood of achieving pCR. In TNBC with RD, both low pre- and post-NAT TILs were associated with shorter RFS. These results suggest that TILs information should be taken into account when additional therapies may be given in the post-neoadjuvant setting.

The role of postmastectomy radiotherapy (PMRT) in breast cancer patients receiving neoadjuvant chemotherapy (NAC) is controversial. We aimed to evaluate the effectiveness of radiotherapy in patients treated with NAC and mastectomy in the Japanese Breast Cancer Registry. We enrolled patients who received NAC and mastectomy for cT1–4 cN0–2 M0 breast cancer. We evaluated the association between radiotherapy and outcomes, locoregional recurrence (LRR), distant disease-free survival (DDFS), and overall survival (OS) based on ypN status by multivariable analysis. Of the 145,530 patients, we identified 3226 who met the inclusion criteria. Among ypN1 patients, no differences were found in LRR, DDFS, or OS between groups with and without radiotherapy (p = 0.72, p = 0.29, and p = 0.36, respectively). Radiotherapy was associated with improved LRR-free survival (p

Gemcitabine (Gem) with paclitaxel (Pac) is used for patients with metastatic breast cancer who require cytoreduction with manageable toxicities. Nanoparticle albumin-bound (nab)-Pac exhibits better efficacy and reduces the risk of hypersensitivity reactions associated with solvent-based Pac. Therefore, Gem plus nab-Pac (GA) therapy may be effective for metastatic breast cancer. The purpose of this study was to determine the maximum tolerated dose for GA therapy.The subjects were patients with metastatic breast cancer with performance status 0 or 1 and normal hepatic, renal and marrow function. Leukopenia, neutropenia or thrombocytopenia of grade 4, neutropenic fever, or non-hematological toxicity of grade 3 or higher during the 1st cycle, and chemotherapy-induced peripheral neurotoxicity of grade 2 or higher at the end of the 1st cycle were defined as dose-limiting toxicities (DLTs). Gem (1250 mg/m(2)) was administered on days 1 and 8. nab-Pac was administered at a starting dose of 180 mg/m(2) (cohort 1) and escalated to 220 mg/m(2) (cohort 2) and 260 mg/m(2) (cohort 3) on day 1 of the 21-day cycle, using a 3 + 3 design.Nine patients (n = 3, 3, and 3 in cohorts 1, 2, and 3, respectively) were included in the study (median age 56 years; range 43-75 years). DLTs did not occur in any cohorts.The initial recommend dose in GA therapy is 1250 mg/m(2) Gem and 260 mg/m(2) nab-Pac. It is well known that nab-Pac has cumulative toxicities, and thus the efficacy and safety of GA therapy require validation in a phase 2 study.

The rate of breast cancer screening for women of all ages in Japan is increasing. However, little is known about the biological differences between screen- and self-detected tumors. We used data from the Japanese Breast Cancer Registry (JBCR), a nationwide registry of newly diagnosed breast cancer cases in Japan, to investigate patients diagnosed between January 1, 2004 and December 31, 2011. We compared the clinicopathological features of tumors and assessed yearly trends regarding the proportion of screen-detected cases during the study period. We found that 31.8 % (65,358/205,544) of cancers were detected by screening. Asymptomatic tumors detected by screening (asymptomatic) were more likely to have favorable prognostic features than those that were self-detected (ductal carcinoma in situ [DCIS]: 19.8 versus 4.1 %, node-negative: 77.0 versus 61.6 %, and estrogen receptor-positive [ER+]: 82.0 versus 72.9 %, respectively). All these findings were statistically significant (p

Background We investigate rates of pathologic complete response (pCR) and tumor expression of ER, PgR, HER2 discordance after neoadjuvant chemotherapy using Japanese breast cancer registry data. Patients and methods Records of more than 300 000 breast cancer cases treated at 800 hospitals from 2004 to 2013 were retrieved from the breast cancer registry. After data cleanup, we included 21 755 patients who received neoadjuvant chemotherapy and had no distant metastases. pCR was defined as no invasive tumor in the breast detected during surgery after neoadjuvant chemotherapy. HER2 overexpression was determined immunohistochemically and/or using fluorescence in situ hybridization. Results pCR was achieved in 5.7% of luminal tumors (n = 8730), 24.6% of HER2-positive tumors (n = 4403), and 18.9% of triple-negative tumors (n = 3660). Among HER2-positive tumors, pCR was achieved in 31.6% of ER-negative tumors (n = 2252), 17.0% of ER-positive ones (n = 2132), 31.4% of patients who received trastuzumab as neoadjuvant chemotherapy (n = 2437), and 16.2% of patients who did not receive trastuzumab (n = 1966). Of the 2811 patients who were HER2-positive before treatment, 601 (21.4%) had HER2-negative tumors after neoadjuvant chemotherapy, whereas 340 (3.4%) of the 9947 patients with HER2-negative tumors before treatment had HER2-positive tumors afterward. Of the 10 973 patients with ER-positive tumors before treatment, 499 (4.6%) had ER-negative tumors after neoadjuvant chemotherapy, whereas 519 (9.3%) of the 5607 patients who were ER-negative before treatment had ER-positive tumors afterward. Conclusion We confirmed that loss of HER2-positive status can occur after neoadjuvant treatment in patients with primary HER2-positive breast cancer. We also confirmed that in practice, differences in pCR rates between breast cancer subtypes are the same as in clinical trials. Our data strongly support the need for retest ER, PgR, HER2 of surgical sample after neoadjuvant therapy in order to accurately determine appropriate use of targeted therapy.

Background: Tumor infiltrating lymphocytes (TIL) in subtypes of breast cancer may provide clinically important information on chemotherapy response and prognosis. However, the standardized methodology for immunohistochemical (IHC)-TIL has not yet been established, reproducible and objective method of evaluation of TIL such as gene expression profiles is warranted. We evaluated whether IHC-TIL level was associated with gene expression profiles and whether such profiles could be used to predict chemotherapy response and prognosis according to subtypes of breast cancers. Methods: To select TIL associated genes, we used 40 samples with both IHC-TIL information and gene expression profiling data. The degree of TIL at the edges of the tumor mass, in the tumor mass, or in the stroma surrounding the expanding mammary ducts packed by carcinoma cells was evaluated as score 0, 1, and 2, when TIL was not unrecognizable (0%), sparse (0 << 50%) and dense (50% ≤), respectively. We selected 22 genes as the TIL-gene signature (GS), by comparing expression profiles between TIL score 2 and 0 tumors. We showed the associations between the TIL-GS levels and subtypes of breast cancers (Estrogen receptor: ER / Human Epidermal growth factor 2: HER2). The chemotherapy sensitivity analysis was performed on cohorts of 625 patients with stage I–III breast cancer who received neo adjuvant chemotherapy (NAC) based on Anthracycline and Taxane containing regimen. Data from 1,586 tumors were used to evaluate the association between distant metastasis free survival (DMFS) and the TIL-GS in a Kaplan-Meier analysis. Results: The TIL-GS for ER negative (-)/HER2- and HER2 positive (+) cases were significantly higher expression level than luminal types (p-value Conclusions: Higher TIL-gene signature of 22 genes appeared to be associated with aggressive subtypes and pCR rate (except luminal-low) of breast cancers. This approach may improve the reproducibility of assessment on tumor TIL level and thus serve the clinical applications for breast cancers. Citation Format: Kochi M, Niikura N, Iwamoto T, Bianchini G, Mizoo T, Nogami T, Shien T, Motoki T, Taira N, Masuda S, Doihara H, Fujiwara T, Tokuda Y, Matsuoka J. Tumor Infiltrating lymphocytes (TIL) related genomic signature associated with chemotherapy response and prognosis in subtypes of breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-14.

Few studies have reported the association between body mass index (BMI) and outcome among Asian breast cancer patients. We analyzed data for 20,090 female invasive breast cancer patients who had been followed‐up for a median period of 6.7 years entered in the National Clinical Database–Breast Cancer Registry between 2004 and 2006. We used mainly the WHO criteria for BMI (kg/m2) categories

Purpose: In spite of improvements of average benefit from adjuvant/neoadjuvant treatments, there are still individual patients with early breast cancer at high risk of relapse. We explored the association with outcome of robust gene cluster–based metagenes linked to proliferation, ER-related genes, and immune response to identify those high-risk patients. Experimental Design: A total of 3,847 publicly available gene-expression profiles were analyzed (untreated, N = 826; tamoxifen-treated, N = 685; chemotherapy-treated, N = 1,150). Genes poorly performing in formalin-fixed samples were removed. Outcomes of interest were pathologic-complete response (pCR) and distant metastasis-free survival (DMFS). In ER+HER2−, the proliferation and ER-related metagenes were combined to define three risk groups. In HER2+ and ER−HER2− risk groups were defined by tertiles of an immune-related metagene. Results: The high-proliferation/low-ER group of ER+HER2− breast cancer had significantly higher pCR rate [OR, 5.01 (1.76–17.99), P = 0.005], but poorer outcome [HR = 3.73 (1.63–8.51), P = 0.0018] than the low-proliferation/high-ER. A similar association with outcome applied to patients with residual disease (RD) after neoadjuvant chemotherapy (P = 0.01). In ER−HER2− and HER2+ breast cancer, immune metagene in the high tertile was linked to higher pCR [33.7% vs. 11.6% in high and low tertile, respectively; OR, 3.87 (1.79–8.95); P = 0.0009]. In ER−HER2−, after adjuvant/neoadjuvant chemotherapy, 5-year DMFS was 85.4% for high-tertile immune metagene, and 43.9% for low tertile. The outcome association was similar in patients with RD (P = 0.0055). In HER2+ breast cancer treated with chemotherapy the association with risk of relapse was not significant. Conclusions: We developed metagene-based predictors able to define low and high risk of relapse after adjuvant/neoadjuvant therapy. High-risk patients so defined should be preferably considered for trials with investigational agents. Clin Cancer Res; 22(2); 337–45. ©2015 AACR.

Background Immune checkpoint therapy only benefits a fraction of patients, thus huge efforts have been made to develop predictive biomarkers to identify those patients. Immune biomarkers like PD-L1 expression are extremely dynamic and the timing of evaluation, on primary or metastatic disease, may be critical. We have already shown that tumour-infiltrating lymphocytes (TILs) decrease during metastatic progression in triple-negative (TN) and human epidermal growth factor-2 positive (HER2+) breast cancers (Ogiya R, ASCO 2015), suggesting that mechanisms of immune escape contribute and favour the metastatic progression. In this work we aimed to characterize the modulation and changes of specific immune markers during the metastatic spread comparing paired samples from primary and recurrent breast cancers. Methods We retrospectively identified 25 patients with HER2+ (n = 14) and TN (n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital, and who subsequently experienced a first regional or distant recurrence confirmed by tumour biopsy/resection. Haematoxylin and eosin-stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was performed using primary antibodies against CD4, CD8, Foxp3, PD-L1, PD-L2, and HLA-class I. Results The sites of first recurrence was the skin (n = 7), brain (n = 6), lymph node (n = 4), lung (n = 3), bone (n = 2), and one of each of bone marrow, liver and muscle. Immunohistochemical evaluations could not be performed in 5 primary tumours and 2 recurrent tumours because of the small quantity of the specimens. The percentage of CD8+ T cells staining in the primary tumours was significantly higher (median 16%) than that in recurrent tumours (median 10%) (paired t-test, p = 0.008) Similarly, the percentage of CD4+ T cells staining in the primary tumours was significantly higher (median 40%) than that in recurrent tumours (median 25%) (p = 0.026). The percentage of Foxp3+ T cells was low ( Comparison of positivity rate between primary and recurrent tumours for each antibody Primary tumourRecurrent tumourPTotal breast tumours (N)2023 TILs positivity rate, median (%) CD440%25%.03CD816%10%.01Foxp3 Conclusions Tumours at first metastatic recurrence in HER2+ and TN breast cancers have a lower percentage of both CD8+ and CD4+ T cells compared to primary tumours, confirming a potential role of immune escape in tumour progression. Other immune markers, including PD-L1, were not found to change significantly, but negative/positive conversions were observed. This suggest that an evaluation of disease at the time of immunotherapy administration might be more informative. These findings warrant larger confirmation studies. Citation Format: Ogiya R, Niikura N, Kumaki N, Bianchini G, Kitano S, Iwamoto T, Hayashi N, Yokoyama K, Oshitanai R, Terao M, Morioka T, Tsuda B, Okamura T, Saito Y, Suzuki Y, Tokuda Y. Difference of immune microenvironment between primary and recurrent tumours in breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-13.

The improvement of tumor biomarkers prepared for clinical use is a long process. A good biomarker should predict not only prognosis but also the response to therapies. In this review, we describe the biomarkers of neoadjuvant/adjuvant chemotherapy for breast cancer, considering different breast cancer subtypes. In hormone receptor (HR)-positive/human epidermal growth factor 2 (HER2)-negative breast cancers, various genomic markers highly associated with proliferation have been tested. Among them, only two genomic signatures, the 21-gene recurrence score and 70-gene signature, have been reported in prospective randomized clinical trials and met the primary endpoint. However, these genomic markers did not suffice in HER2-positive and triple-negative (TN) breast cancers, which present only classical clinical and pathological information (tumor size, nodal or distant metastatic status) for decision making in the adjuvant setting in daily clinic. Recently, patients with residual invasive cancer after neoadjuvant chemotherapy are at a high-risk of recurrence for metastasis, which, in turn, make these patients best applicants for clinical trials. Two clinical trials have shown improved outcomes with post-operative capecitabine and ado-trastuzumab emtansine treatment in patients with either TN or HER2-positive breast cancer, respectively, who had residual disease after neoadjuvant chemotherapy. Furthermore, tumor-infiltrating lymphocytes (TILs) have been reported to have a predictive value for prognosis and response to chemotherapy from the retrospective analyses. So far, TILs have to not be used to either withhold or prescribe chemotherapy based on the absence of standardized evaluation guidelines and confirmed information. To overcome the low reproducibility of evaluations of TILs, gene signatures or digital image analysis and machine learning algorithms with artificial intelligence may be useful for standardization of assessment for TILs in the future.

Background: Fulvestrant is one of the standard treatments for first- and second-line endocrine therapy for hormone receptor (HR)-positive metastatic breast cancer (MBC). Palbociclib inhibits cyclin dependent kinase (CDK) 4 and CDK6 in vitro, resulting in loss of RB1 phosphorylation. Palbociclib has high activity in HR-positive breast cancer cell lines and is synergistic in combination with endocrine therapies. The PALOMA-3 trial enrolled patients with metastatic estrogen receptor (ER)-positive breast cancer with resistance to aromatase inhibitor. The results demonstrated that palbociclib combined with fulvestrant was associated with significantly longer progression-free survival (PFS) than fulvestrant alone. However, patients in the trial reported higher rates of adverse events, such as neutropenia and alopecia, compared with fulvestrant monotherapy. We investigate in this prospective cohort study that the addition of palbociclib to fulvestrant is effective and safe in HR-positive and HER2-negative advanced breast cancers that progressed during fulvestrant monotherapy. Specific Aims: Primary objective: To observe the PFS in patients with resistance to fulvestrant monotherapy who were treated with fulvestrant plus palbociclib. Secondary Objective: To observe the PFS in patients treated with fulvestrant as the first and second line therapy. We also aim to observe overall survival and safety. A prospective translational research is also planned to assess the correlations between biomarkers and response. Trial Design: Single arm exploratory trial Eligibility Criteria: Eligible patients must have historically confirmed ER-positive and HER2-negative MBC who received fulvestrant as first or second line therapy. Prior chemotherapy in the neoadjuvant and adjuvant settings and up to one line of chemotherapy in MBC is permissible. Patients who received CDK4/6 inhibitor as metastatic therapy are excluded. Statistical Design: We aim to set the lowest limit of the therapeutic effect at a median PFS of 5 months, which is the value reported in the PALOMA-3 study. If we assume that the expected median PFS is 8 months, at least 63 patients are required to reject the null hypothesis (5 months) with a power of 80% under a one-sided alpha at 0.05. The length of the accrual period and follow-up period are set at 12 and 18 months, respectively. Target Accrual: First registration: 200 patients with fulvestrant monotherapy as the first and second line setting. Second registration: 70 patients with combined palbociclib and fulvestrant. (UMIN 000029294) Citation Format: Kokoro Kobayashi, Naoki Niikura, Shigehira Saji, Takayuki Iwamoto, Nobutaka Iwakuma, Yuichiro Kikawa, Norikazu Masuda, Kenichi Watanabe, Takashi Takeshita, Mari Oba, Shinji Ohno. Fulvestrant with additional palbociclib in advanced or metastatic hormone receptor-positive HER2-negative breast cancer after progression to fulvestrant monotherapy: JBCRG- M07 (FUTURE trial) [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 OT2-02-07.

A population-based cancer registry has been used for the planning and evaluation of cancer control activities based on administration and the care of individual cancer patients by those in the medical profession. The Japanese Breast Cancer Society (JBCS) registry was started in 1975. In 2004, the registry system was moved to a new system using web registration with the cooperation of the Non-Profit Organization Japan Clinical Research Support Unit and Public Health Research Foundation (Tokyo, Japan). Comprehensive individual patient data were recorded according to the Unio Internationalis Contra Cancrum (UICC) TNM classification [1] and the World Health Organization histological classification [2]. The details are described elsewhere [3]. Annual reports on this registry have since been published in Japanese and publicized through the JBCS web site to active members of the JBCS [4]. We herein report the results of a 5-year prognostic analysis of cases registered in 2005 (Figs. 1, 2, 3, 4, 5, 6, 7, 8 and 9; Supplementary Tables 1–9). The number of facilities involved in the 2005 registration was 354 and the total number of cases was 20,786. The estimated incidence of breast cancer was reported to be 50,695 cases in 2005 by the National Cancer Center [5]. Therefore, approximately Electronic supplementary material The online version of this article (doi:10.1007/s12282-015-0645-4) contains supplementary material, which is available to authorized users.

Background The aims of this study were to determine clinicopathological factors associated with postoperative upstaging to invasive carcinoma in patients preoperatively diagnosed with ductal carcinoma in situ (DCIS) and to develop a model to predict the risk of upstaging. Methods Pre- and post-operative pathological diagnoses and radiological findings were assessed for 1,187 consecutive patients. Results Of the patients, 306 (25.8%) were upstaged on the surgical specimen. In multivariate analysis, the following four factors were significantly associated with upstaging: 1) the presence of sclerosing adenosis on the preoperative biopsy specimen (odds ratio [OR] 0.46, P = 0.013); 2) pleomorphic calcifications on the mammogram (OR 1.68, P = 0.009); 3) a mass suspicious for invasive carcinoma on ultrasonography and/or MRI (OR 2.13, P

We prospectively compared the diagnostic accuracies of PET/CT and BS in patients with suspected bone metastases from breast cancer. This single-institution prospective study included consecutive patients with suspected bone metastases from biopsy-proven breast cancer seen at Tokai University Hospital between September 2011 and March 2014. Inclusion criteria included suspicions for bone metastases (bone pain, elevated alkaline phosphatase, elevated tumor markers, or suspected bone metastases by BS). Two nuclear medicine physicians evaluated PET/CT and BS images. Thirty patients were initially enrolled in this study. Two were excluded from the analyses because they declined to undergo imaging during follow-up. PET/CT successfully detected bone metastases in all 10 patients finally diagnosed with the condition, whereas BS identified 2. The two methods were not highly concordant in detecting osseous metastases. In 19 of 28 paired studies (68 %), 2 (10 %) were positive for metastasis, and 17 (90 %) were negative. Nine occurrences (32 %) were discordant; of these, 2 were PET/CT positive and BS negative; 5 were PET/CT positive and BS equivocal; one was PET/CT negative and BS equivocal, and one was PET/CT equivocal and BS negative. Our results indicated that PET/CT was superior to BS for the diagnosis of bone metastases. On the basis of the results of previous studies as well as ours, PET/CT could replace BS as the initial modality to detect bone metastases in patients suspected for the condition.

A 55-year-old woman underwent a total thyroidectomy for carcinoma showing thymus-like differentiation (CASTLE). The patient was referred to our hospital after the tumor was found to have directly invaded the cervical esophagus and the entire circumference of the trachea. A total thyroidectomy was performed, followed by end-to-end anastomosis of the trachea, suprahyoid release and dissection of bilateral pulmonary ligaments. No major complications, including anastomotic dehiscence or stenosis, were observed. The patient experienced some swallowing disturbances and hoarseness during the perioperative period but fully recovered. Radiotherapy to the neck was performed as an adjuvant therapy. Eleven months after surgery, lower back pain and right leg numbness developed and led to gait inability. Multiple lung and bone recurrences were observed, but no local recurrence. Palliative radiotherapy to the bone metastasis was performed. The patient died of pleural metastasis 14 months after the initial diagnosis of CASTLE.

A lack of consistent methods to evaluate Ki67 expression is problematic in terms of accurately predicting prognosis in breast cancer. Accordingly, this study aimed to identify the causes of discrepancies in Ki67 labeling index measurements by different observers under different conditions using breast cancer samples.This Japanese study group compared and assessed immunohistochemical (IHC) analysis of the Ki67 labeling index when measured by different pathologists. Six pathologists (pathologists A-F) in Japan participated in this ring study. One hundred and ten surgical cases of estrogen receptor-positive and human epidermal growth factor receptor 2-negative invasive breast cancer treated in 2007 were identified from the breast cancer database of Tokai University Hospital and were included in this study.For all 6 pathologists, the Ki67 labeling index were significantly different between grade 3 and grade 1 cases and between grade 3 and grade 2 cases, whereas the index tended to be different between grade 1 and grade 2 cases. Further, the Ki67 labeling indexes measured by the 6 pathologists were strongly correlated (ρ: 0.73-0.88). The IHC scores recorded by pathologist A were in moderate to good agreement with those recorded by the others in patients with a Ki67 labeling index of13.25 % and in those with a Ki67 labeling index of13.25 % (κ = 0.429-0.660). The Ki67 low and high concordance rates between pathologist A and the others were 0.452-0.778 and 0.862-0.979, respectively. The most pertinent reason for discrepancy in scores seemed to be the selection of the area for counting and the quality of nuclear staining.The Ki67 labeling index measured by 6 pathologists without method standardization was in fair to good agreement. We plan to undertake a second ring study, pending recommendations by the international Ki67 panel.

Breast cancers (BC) carry a complex set of gene mutations that can influence their gene expression and clinical behavior. We aimed to identify genes driven by the TP53 mutation status and assess their clinical relevance in estrogen receptor (ER)-positive and ER-negative BC, and their potential as targets for patients with TP53 mutated tumors. Separate ROC analyses of each gene expression according to TP53 mutation status were performed. The prognostic value of genes with the highest AUC were assessed in a large dataset of untreated, and neoadjuvant chemotherapy treated patients. The mitotic checkpoint gene MPS1 was the most significant gene correlated with TP53 status, and the most significant prognostic marker in all ER-positive BC datasets. MPS1 retained its prognostic value independently from the type of treatment administered. The biological functions of MPS1 were investigated in different BC cell lines. We also assessed the effects of a potent small molecule inhibitor of MPS1, SP600125, alone and in combination with chemotherapy. Consistent with the gene expression profiling and siRNA assays, the inhibition of MPS1 by SP600125 led to a reduction in cell viability and a significant increase in cell death, selectively in TP53-mutated BC cells. Furthermore, the chemical inhibition of MPS1 sensitized BC cells to conventional chemotherapy, particularly taxanes. Our results collectively demonstrate that TP53-correlated kinase MPS1, is a potential therapeutic target in BC patients with TP53 mutated tumors, and that SP600125 warrant further development in future clinical trials.

Introduction: The one-step nucleic acid amplification (OSNA) assay is a new method to detect sentinel lymph node (SLN) metastases using cytokeratin 19 (CK19) mRNA in early breast cancer. Here we retrospectively analyzed the advantages and disadvantages of the OSNA assay. Methods: In a trial period, SLNs were divided into two sections, and we examined one side using the OSNA assay. The other side was examined by pathologists. After this period, we examined whole SLNs using only the OSNA assay. The patients with positive nodes by OSNA assay and/or pathology required axillary dissection. Results: We examined 27 primary breast cancer patients (36 SLNs) during the trial period. The overall concordance rate between the OSNA assay and pathology results was 91%. In the later period, 157 patients (217 SLNs) were examined. The CK19-positive rate obtained by the OSNA assay was 16.5% (macrometastases OSNA (++) : 7.2%, micrometastases OSNA (+) : 9.2%). The non-SLN positive rate among the CK19-positivecases was 23%. The OSNA assay's false negative was one case in which the expression of CK-19 on the primary tumor and lymph node was not detected. Conclusions: Our OSNA assay results were comparable to those obtained using a conventional pathological technique. Pathologists and laboratory technicians could save time and effort by using the OSNA assay when seeking the precise diagnosis during surgery.

e14182 Background: Metformin is one of the most commonly prescribed drugs for type 2 diabetes, and some reports have suggested that metformin may reduce cancer risk. Diabetics treated with metformin have a 23% reduction in the risk of cancer, including breast cancer. In addition, it is reported that the breast cancer patients with metformin treatment for diabetes showed favorable prognosis compared with those without metformin treatment. However, the mechanism underlying the positive effects of metformin on cancer treatment remains unclear. Methods: We conducted a prospective study to evaluate the effect of preoperative metformin on early breast cancer patients. The patients took a daily dose of metformin orally for 7 to 21 days before surgery. We evaluated the effects on immunological factors (TILs, CD4+, CD8+, PD-L1 and ALDH1) by comparing core needle biopsies (CNB) obtained before surgery with surgical specimens. Results: Seventeen breast cancer patients were enrolled in this prospective study and administered metformin before surgery, during the period from January to December 2016. We analyzed 59 patients who received surgery during the same period as a control group. In the control group, there was no significant difference in TILs between CNB and surgical specimens (Rs = 0.63). In the metformin group, TILs were negative in CNB and surgical specimens of 15 (88%) and 8 (48%) cases, low in 2 (12%) and 8 (48%), and intermediate in 0 and 1 (6%), respectively. These TILs increases were confirmed in 5 (29%) patients (p = 0.09), while a decrease was confirmed in 2 (12%). The expressions of CD4+ and CD8+ by TILs were increased in 41% and 18% of surgical specimens, respectively. (p = 0.02, p = 0.09) However, there was no statistically significant difference in these immunological factors and PD-L1 or ALDH1 expression between before and after metformin. Conclusions: In our small cohort, preoperative metformin administration shows positive impact on CD4 positive lymphocytes significantly and has tendency of increasing both TILs and CD8 positive lymphocytes. However, we should keep in mind that our sample size is small. Further study is necessary to uncover the mechanisms of favorable effects of metformin on breast cancer patients.

[Background] Based on the results of a case-control study performed in Japan (Okayama and Kagawa), it was revealed that gene polymorphisms of the estrogen receptor (single nucleotide polymorphisms, SNPs), ESR1/6q25.1-rs2046210 and SNPs of rs3757318 were closely related to breast cancer risk in Japanese women, and that the odds ratio per allele of individual SNPs were 1.37 [95% CI: 1.1 - 1.7] and 1.33 [1.04 - 1.7], respectively (San Antonio Breast Cancer Symposium 2012, Mizoo et al.). We analyzed the relationship of ESR1/6q25.1-rs2046210 and rs3757318 SNPs with the clinical characteristics of breast cancer patients. [Methods] Among the patients enrolled in the case-control study, 508 breast cancer patients who underwent genotyping for ESR1/6q25.1-rs2046210 and rs3757318 were analyzed as the subjects. SNPs were analyzed using the TaqMan genotyping assay® for blood samples. To analyze the relationship of genotypes (AA/GG, GG) with age at diagnosis, height, weight, BMI, age of menarche, birth history, pathological degree of progression and estrogen receptor, t-test and chi-square test were used. [Results] The genotypes of rs2046210 are AA (12%), AG (41%) and GG (46%), and the patients with the risk allele (AA/AG) had significantly higher height (p = 0.0015, t-test) and lower Body Mass Index (BMI) (p = 0.0022, t-test). The genotypes of rs3757318 are AA (8%), AG (39%) and GG (53%), and the patients with the risk allele (AA/AG) were characterized by significantly lower BMI (p = 0.041, t-test), no birth history (p = 0.0473, chi-square) and negative hormone receptor (p = 0.0128, chi-square). Overall, tendencies related to increased height, lower BMI and negative estrogen receptor were confirmed for the risk alleles of rs2046210 and rs3757318. [Conclusion] It was suggested that SNPs of rs2046210 and rs3757318 might be related to the development of estrogen-receptor-negative breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-10.

Background The prognostic and predictive relevance of different components of the adaptive immune system in HER2-positive, TN and high proliferation ER+/HER2- subtypes have been shown. In this scenario, the remarkable therapeutic efficacy of new immunomodulatory drugs targeting the immune checkpoints (i.e. CTLA4) provides attractive opportunities. We aimed to investigate the prognostic value of genes related to CD28 and CTLA-4 signaling pathways in early breast cancer patients. Methods We assessed the affymetrix gene expression data of 789 untreated, node-negative, early breast cancer patients. Expression of CTLA4, CD28 and the two corresponding natural ligands CD80 (B7-1) and CD86 (B7-2) were evaluated. The sum of these two ligands was combined as a unique marker (CDligs). The prognostic value of the three biomarkers and their interaction were assessed independently in each molecular subtype (ER-/HER2-, HER2+ and high proliferation ER+/HER2- [ER+/HER2- HP]). Multivariate analysis was adjusted for PGR, proliferation and other immune related genes (Tcell, IGG, MHC II, MHC I, Interferon and STAT1) (Gianni L, SABCS 2012). Distant event free survival was the outcome of interest. Results A significant moderately positive correlation was observed between each biomarker. Expression of CTLA4 and CDligs was higher in ER-/HER2- and HER2+ compared to luminal subtypes. High expression of CTLA4 and CD28 were associated with lower risk of relapse in HER2+ (p = 0.008 and p = 0.03, respectively) and ER+/HER2-HP (p = 0.03 and p = 0.008, respectively). High expression of CDligs was also associated with lower risk of distant event in HER2+ group only (p = 0.01). No interactions were observed between CD28 and CTLA4 in any subtype. In both TN and ER+/HER2- HP subtypes, a significant interaction between CDligs and CTLA4 (interaction term p = 0.009 and p = 0.025, respectively), CDligs and CD28 (interaction term p = 0.028 and p = 0.008, respectively) and CDligs and Tcell metagene (interaction term p = 0.006 and p = 0.019, respectively) was observed. For instance, in high-CTLA4 group (above median cut point), higher expression of CDligs was associated with significantly lower risk of distant relapse in TN [HR 0.41 (0.21-0.82); p = 0.012], HER2+ [HR 0.25 (0.08-0.72); p = 0.011) and ER+/HER2- HP [HR 0.40 (0.29-0.85); p = 0.01] subtypes. Instead, in the low-CTLA4 group, CDligs was not associated with distant relapse in any subtype [TN, HR 1.80 (0.28-3.96) p = 0.141; HER2+, HR 0.69 (0.24-1.99) p = 0.495; ER+/HER2- HP, HR 1.28 (0.70-2.35) p = 0.425]. These interactions retain statistical significance in multivariate analysis including PGR, proliferation scores and other immune metagenes. Conclusions Genes related to the CD28/CTLA4 immune checkpoint are associated with risk of distant relapse independently of other biomarkers and immune related metagenes. A significant interaction between expression of ligands (CD80/CD86) and the two corresponding receptors CD28 and CTLA4 was found in TN and ER+/HER2- HP subtypes. These data confirm the complex interactions involved in the adaptive immune system regulation which may not be fully appreciated without consideration of biomarker interactions. They also provide additional evidence for exploring the use of immunomodulatory drugs in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-04.