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Herein, we report a case of a patient with recurrent breast cancer who was diagnosed antemortem with pulmonary tumor thrombotic microangiopathy (PTTM) using wedge aspiration cytology of the pulmonary artery after breast cancer surgery.The patient was a 50-year-old woman who underwent mastectomy and axillary lymph node dissection for stage IIIA (T3N2M0) triple-negative left breast cancer. Postoperative follow-up was performed with radiotherapy and anticancer chemotherapy. Seventeen months after the surgery, the patient was hospitalized for right heart failure and diagnosed with pulmonary arterial hypertension. The patient was diagnosed with PTTM following the detection of malignant cells in the pulmonary artery using wedge aspiration cytology. Anti-pulmonary hypertension therapy was administered; however, the patient did not respond and died 26 days after admission. Autopsy revealed multiple microscopic tumor emboli in the pulmonary artery. In portions of the pulmonary artery without embolization, fibro-cellular intimal hyperplasia and stenosis were observed. Tumor embolism was expressed for CK7+/CK20-, consistent with the primary breast cancer.Since the primary pathophysiology of PTTM entails narrowing due to fibro-cellular intimal hyperplasia rather than multiple tumor thrombi, the efficacy of chemotherapy combined with vasodilators is discussed.

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

Background Historically, humoral immunity was considered unimportant in anti-tumor immunity, and the differentiation and anti-tumor activity of B cells in breast cancer are poorly understood. However, it was recently discovered that B cells participate in tumor immunity through both antibody production and immunosuppressive mechanisms. We analyzed the expression of B-cell differentiation markers in detail using fluorescence-activated cell sorting to investigate the relationship between B-cell subsets and breast cancer etiology. Methods Blood samples were taken from breast cancer patients and healthy donors, and peripheral blood mononuclear cells were collected. B cells at various stages of differentiation were identified by the expression of combinations of the cell surface markers CD5, CD19, CD21, CD24, CD27, CD38, CD45, and IgD. Statistical analysis of the proportions of each B-cell subtype in the different patient groups was then performed. Results Twenty-seven breast cancer patients and 12 controls were considered. The proportion of total B cells was significantly higher in cancer patients than in controls (11.51 ± 2.059 vs 8.905 ± 0.379%, respectively; p = 0.001). Breast cancer patients were then classified as High-B or Low-B for further analysis. A significantly higher proportion of memory B cells was found in the High-B group than in the Low-B or control groups (p = 0.003 and p = 0.043, respectively). Conclusions Breast cancer patients generally have a higher proportion of B cells than healthy controls, but this is highly variable. Analysis of the major B-cell surface markers indicates that memory B cells in particular are significantly expanded, or more robust, in breast cancer patients. Electronic supplementary material The online version of this article (10.1007/s12282-017-0824-6) contains supplementary material, which is available to authorized users.

// Rin Ogiya 1 , Naoki Niikura 1 , Nobue Kumaki 2 , Hiroyuki Yasojima 3 , Tsutomu Iwasa 4 , Chizuko Kanbayashi 5 , Risa Oshitanai 1 , Michiko Tsuneizumi 6 , Ken-ichi Watanabe 7 , Akira Matsui 8 , Tomomi Fujisawa 9 , Shigehira Saji 10 , Norikazu Masuda 3 , Yutaka Tokuda 1 and Hiroji Iwata 11 1 Department of Breast and Endocrine Surgery, Tokai University School of Medicine, Kanagawa, Japan 2 Department of Pathology, Tokai University School of Medicine, Kanagawa, Japan 3 Department of Surgery, Breast Oncology, National Hospital Organization Osaka National Hospital, Osaka, Japan 4 Department of Medical Oncology, Kindai University School of Medicine, Osaka, Japan 5 Department of Breast Oncology, Niigata Cancer Center Hospital, Niigata, Japan 6 Department of Breast Surgery, Shizuoka General Hospital, Shizuoka, Japan 7 Department of Breast Surgery, Hokkaido Cancer Center, Sapporo, Japan 8 Department of Surgery, National Hospital Organization, Tokyo Medical Center, Tokyo, Japan 9 Department of Breast Oncology, Gunma Prefectural Cancer Center, Gunma, Japan 10 Department of Medical Oncology, Fukushima Medical University, Fukushima, Japan 11 Department of Breast Oncology, Aichi Cancer Center Hospital, Nagoya, Japan Correspondence to: Naoki Niikura, email: niikura@is.icc.u-tokai.ac.jp Keywords: breast cancer, brain metastases, tumor infiltrating lymphocytes, PD-L1, immune microenvironment Received: July 11, 2017 Accepted: September 20, 2017 Published: October 27, 2017 ABSTRACT Background: Immune checkpoint inhibitors are reported to be effective in patients with brain metastases. However, detailed characteristics of the brain metastasis immune microenvironment remain unexplored. Results: The median tumor-infiltrating lymphocyte (TIL) category in brain metastases was 5% (1–70%). In 46 pair-matched samples, the percentages of TILs were significantly higher in primary breast tumors than in brain metastases (paired t -test, P < 0.01). The numbers of CD4/CD8/Foxp3-positive cells were significantly higher in primary breast tumors than in brain metastases (paired t -test, P < 0.05 for all antibodies). In patients with triple-negative breast cancer specifically, low TIL numbers were associated with significantly shorter overall survival compared to high TIL numbers (log-rank test, P = 0.04). Materials and Methods: We retrospectively identified 107 patients with breast cancer and brain metastases who had undergone surgery between 2001 and 2012 at 8 institutions, and collected 191 samples including brain metastases alone and primary tumors with pair-matched brain metastasis samples. Hematoxylin and eosin-stained slides were evaluated for TILs and categorized according to the extent of staining. Immunohistochemistry for CD4, CD8, Foxp3, PD-L1, PD-L2, and HLA class I was also performed. Conclusions: There are significantly fewer TILs in brain metastases than in primary breast tumors.

Introduction Axillary lymph node dissection is standard therapy for patients with positive-node breast cancer, and can be performed with an electrocautery scalpel and suture ligation in most cases. However, knot slipping can occur during suture ligation and this can spread thermal damage to peripheral tissues. The LigaSureTM Small Jaw vessel-sealing system was developed as an alternative to suture ligatures, staplers, and other energy-dependent devices for sealing blood and lymphatic vessels, but its use in axillary dissection for breast cancer is limited. We prospectively compared the duration until drain removal after surgery, total lymph fluid drainage volume, intraoperative blood loss, and incidence of complications after axillary dissections, between this device and conventional methods. Methods This prospective randomized study was conducted at the Department of Breast and Endocrine Surgery at Tokai University School of Medicine, Kanagawa, Japan, between October 2011 and March 2015. Major eligibility criteria included (1) pathologically confirmed breast cancer diagnosis, (2) age ≥20 and ≤80 years, and (3) a signed informed consent form. The primary endpoint was duration until drain removal after surgery. The secondary endpoints were total lymph fluid drainage volume, intraoperative blood loss, and incidence of postoperative surgical complications. We defined the criterion for drain removal as a lymph fluid drainage volume of Results Initially, 100 patients were assigned as eligible; however, two patients were later excluded because of the exclusion criteria. Of 98 patients, 49 were randomized to the study group, and 49 to the control group. The mean duration until drain removal after surgery was 5.2 days in the study group and 5.0 days in the control group (p=0.573). The mean total lymph fluid drainage volumes were 260.3 and 233.5 mL (p=0.502), and the mean intraoperative blood loss volumes were 17.8 and 18.0 mL (p=0.949), for the study and control groups, respectively. No significant differences were found between the two groups regarding drain removal duration, total drainage volume, and intraoperative blood loss volume. Both groups had low incidence rates of postoperative hematoma, wound infection, lymphedema, and pain, and had similar incidence rates of seroma formation after drain removal. Conclusion Our study results indicated that the use of the LigaSureTM Small Jaw in axillary dissection for breast cancer was as safe as conventional methods. However, using the LigaSureTM Small Jaw did not improve surgical outcomes such as duration until drain removal and total lymph fluid drainage volume compared with conventional methods. Citation Format: Okamura T, Niikura N, Yokoyama K, Ogiya R, Oshitanai R, Terao M, Morioka T, Tsuda B, Saito Y, Suzuki Y, Tokuda Y. Utility of LigaSureTM vessel-sealing device in axillary dissection for breast cancer surgery: A randomized single center study [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 P3-13-11.

Background Immunoglobulin (Ig) G4-related sclerosing disease is a pathological concept proposed in Japan during the early 2000s. This lesion-forming disease may exhibit characteristics of a systemic disease but often affects a single organ. To date, IgG4-related sclerosing disease in the mammary gland, or IgG4-related mastitis (IgG4-RM), has rarely been reported. Case presentation Here, we describe the case of a female patient who was admitted to our hospital with the main complaints of left breast and axillary lymphadenopathy. A careful diagnostic imaging examination led to an initial suspicion of breast cancer. However, a needle biopsy led to a diagnosis of IgG4-RM. Subsequently, the patient was successfully treated with predonin. Conclusions The treatment requirements for breast cancer and IgG4-RM differ considerably. This is a good example of a case wherein unnecessary surgical treatment, which is indicated for breast cancer, was avoided by needle biopsy. Accordingly, the patient was appropriately treated with steroids following a correct diagnosis.

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.

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.

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.

Background: HER2-positive breast cancer has a high risk of developing brain metastasis compared to other subtypes of breast cancer. However, the clinical course and prognostic factors of HER2-positive breast cancer patients with brain metastases are not well known because of the relatively small population. The aim of this study was to determine clinicopathological factors associated with prognosis of HER2-positive patients developed brain metastasis. Methods: A retrospective large dataset of 432 HER2-positive patients who were diagnosed with brain metastases between 2001 and 2012 were collected from 24 institutions of the Japan Clinical Oncology Group: Breast Cancer Study Group. We assessed the clinicopathological factors associated with prognosis of these populations with brain metastases. Results: The median age of the 432 patients was 54 years (range, 20–86 years). Of the patients, 162 patients (37.5%) had ER-positive/HER2-positive (ER+HER2+) breast cancer and 270 patients (62.5%) had ER-negative/HER2-positive (ER-HER2+) breast cancer. Nineteen of the 162 patients with ER+HER2+ (12%) and 53 of the 270 patients with ER-HER2+ (20%) underwent surgery for brain metastases. After the diagnosis of brain metastasis, 108 patients with ER+HER2+ (63%) and 175 patients with ER-HER2+ (64%) received HER2-targeting agents, including trastsuzumab and/or lapatinib. The median brain metastasis-free survival period from the diagnosis of primary breast cancer was 33.5 month in both subtypes. In 63.4% of patients with ER+HER2+subtype and 75.6% of patients with ER-HER2+, brain metastases were detected within 2 years after development of first distant metastasis. Eighty-four patients with ER+HER2+ subtype (52%) and 133 patients with ER-HER2+ (49%) had more than 3 brain metastases at the diagnosis. The median survival period after developing brain metastasis was 16.5 months (95% confidence interval [CI], 11.9–21.1 months) in patients with ER+HER2+ and 11.5 months (95% CI, 9.1–13.8 months) in patients with ER-HER2+ (p = 0.117). Patients with more than 3 brain metastases had significantly shorter OS period than patients with equal or less than 3 brain metastases in both of ER+HER2+ (p < 0.001) and ER-HER2+ (p = 0.018). According to receiving HER2-targeting agents, patients receiving both of trastsuzumab and lapatinib had significantly longer survival period than patients who had received trastsuzumab alone, lapatinib alone, or no HER2-targeting agent (p < 0.001). Conclusions: Our results showed that HER2-positive patients with more than 3 brain metastases at the diagnosis had poor prognosis regardless of ER-positivity, and receiving both of trastsuzumab and lapatinib might improve their survival. Further studies are needed to determine the best treatment strategy including these HER2-targeting agents for these populations. Citation Format: Naoki Hayashi, Naoki Niikura, Norikazu Masuda, Seiki Takashima, Rikiya Nakamura, Ken-Ichi Watanabe, Chizuko Kanbayashi, Mayumi Ishida, Yasuo Hozumi, Michiko Tsuneizumi, Naoto Kondo, Yoichi Naito, Yayoi Honda, Akira Matsui, Tomomi Fujisawa, Risa Oshitanai, Hiroyuki Yasojima, Hideko Yamauchi, Shigehira Saji, Hiroji Iwata. Prognostic factor of HER2-positive breast cancer patients developed brain metastasis: A multicenter retrospective analysis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-16-08.

The clinical course and prognostic factors of HER2-positive breast cancer patients with brain metastases are not well known because of the relatively small population. The aim of this study was to determine prognostic factors associated with HER2-positive patients who develop brain metastases. This retrospective study assessed the largest dataset to date of 432 HER2-positive patients who were diagnosed with brain metastases from 24 institutions of the Japan Clinical Oncology Group, Breast Cancer Study Group. The median age of the 432 patients was 54 years (range, 20–86 years). Of the patients, 162 patients (37.5 %) had ER-positive/HER2-positive (ER+HER2+) breast cancer, and 270 (62.5 %) had ER-negative/HER2-positive (ER−HER2+) breast cancer. The median brain metastasis-free survival period from primary breast cancer was 33.5 months in both groups. The median survival after developing brain metastasis was 16.5 and 11.5 months in the ER+HER2+ and ER−HER2+ groups, respectively, (p = 0.117). Patients with >3 brain metastases had significantly shorter overall survival in both ER+HER2+ (p < 0.001) and ER−HER2+ (p = 0.018) groups. Treatment with trastuzumab before developing brain metastases was not associated with survival duration after developing brain metastases (p = 0.571). However, patients treated with both trastuzumab and lapatinib after developing metastasis had significantly longer survival than patients treated with trastuzumab alone, lapatinib alone, or no HER2-targeting agent (p < 0.001). For HER2-positive patients with brain metastases, regardless of the use of trastuzumab before developing brain metastasis, treatment with both trastuzumab and lapatinib might improve survival.

To define prognostic factors for breast cancer patients with brain metastases, compare their clinical courses and prognoses according to breast cancer subtypes, and analyze the causes of death in such patients. We retrospectively analyzed 1,466 patients diagnosed with brain metastases between April 1, 2001 and December 31, 2012, from 24 institutions of the Japan Clinical Oncology Group. Overall, 1,256 patients with brain metastases were included. The median overall survival (OS) was 8.7 months (95 % confidence interval [CI] 7.8–9.6 months). Univariate and multivariate analyses revealed that patients diagnosed with brain metastasis within 6 months of metastatic breast cancer diagnoses, asymptomatic brain disease, or HER2-positive/estrogen receptor-positive tumors had increased OS. Median OS after the development of brain metastases was 9.3 months (95 % CI 7.2–11.3) for the luminal type, 16.5 months (95 % CI 11.9–21.1) for the luminal-HER2 type, 11.5 months (95 % CI 9.1–13.8) for the HER2 type, and 4.9 months (95 % CI 3.9–5.9) for the triple-negative type. Luminal-HER2 type patients had significantly longer OS than patients with the luminal type (hazard ratio [HR] = 1.50, P

We report a patient with a giant phyllodes tumor of the right breast associated with a hypoglycemic attack. A 48-year-old woman experienced a loss of consciousness and was transferred via ambulance to our hospital emergency department. Upon arrival, her blood glucose level was 26 mg/dl, and a giant tumor (20 cm in diameter) with skin ulceration was observed on the right breast. Core needle biopsy led to a histological diagnosis of a phyllodes tumor of the breast. Ultrasonography and computed tomography detected neither distant metastasis nor a pancreatic endocrine tumor. Her preoperative serum insulin-like growth factor (IGF)-II and insulin levels were 1,330 ng/ml (normal range, 519-1067 ng/ml) and1.0 µU/ml, respectively. Following a simple mastectomy, the 24-h postoperative serum IGF-II and insulin levels were 496 ng/ml and 10.0 µU/ml, respectively. The IGF-II levels detected in the phyllodes tumor and normal breast tissue were 10,600 ng/Wg (wet weight in grams) and 855 ng/Wg. We conclude from these findings that the hypoglycemic attack was related to the elevated IGF-II level in the giant phyllodes tumor of the breast.

Background: Bisphosphonate therapy has decreased the risk of skeletal complications associated with osteolytic bone lesions in patients with breast cancer and multiple myeloma. The large prospective studies have used 21 to 24 months of treatment. We studied the safety and efficacy of Zoledronic acid in a subset of patients who received therapy for more than 24 months. Patients and Methods: Patients who received Zoledronic acid were identified. Data on skeletal events and laboratory parameters were gathered by chart review. The treatment regimen is 4 mg of Zolendronic acid at 3- to 4-week intervals, and concurrent chemotherapy, hormonal therapy, and radiation therapy was included. Before approval of Zoledronic acid, we were using Pamidronate and Incadronate. Results: We used the Zoledronic acid in 221 patients from June 2006 until December 2011 (range, 1–69 times of administration). We analyzed 71 cases in which the treatment could be continued for more than 24 months (range, 24–69 times of administration). No significant calcium, phosphorus, electrolyte abnormalities were encountered. There were no significant differences between the long-term treatment patients with Zoledronic acid and all the other patients in the range of pain felt at the time of the bone metastasis diagnosis, or the bone metastases sites. In addition, by March 2012, more than 50% patients of long-term usage of Zoledronic acid had continued receiving treatment. And in all of the patients, as well, the most cited reason for discontinuing treatment was a disease progression, adverse effect was few. In long-term treatment patients, 4 cases of fractures and 2 cases of spinal compression were encountered. The median time until an SRE occurred was 37 months. There were fewer occurrences of SREs in our investigation than in the 12 months of a clinical trial conducted in Japan with Zoledronic acid and a placebo. As for adverse effects, BRONJ appeared in 4 (1.8%) out of a total of 221 cases, and in 3 (4.2%) at prolonged treatment patients. The other adverse effects were fever in 4 cases and fatigue in 2 cases. Conclusion: Prolonged treatment with Zoledronic acid seems to be well tolerated and should be studied in prospective, randomized studies to document prolonged skeletal efficacy and survival. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-13-02.

Objective: The purpose of this study was to assess the safety of S-1 in Japanese in inoperable or recurrent breast cancer patients. Methods: A prospective post-marketing surveillance was performed at 313 sites in Japan in patients with inoperable or recurrent breast cancer treated with S-1. We examined 1361 patients between January 2006 and December 2007 with regard to the incidence of adverse drug reactions graded by the Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. Results: At least one adverse drug reaction was encountered by 858 patients, with an overall incidence of 63.0% (858/1361). The incidence of Grade 3 or higher adverse drug reactions in a descending order was 14.7% (200/1361). In this study, the most common combination drug was trastuzumab. The overall incidence of adverse drug reactions was 63.5% (431/679 patients) in patients treated with S-1 alone, and 55.9% (66/118 patients) in patients treated with S-1 þ trastuzumab. Conclusions: Monotherapy with S-1 or combination therapy with S-1 þ trastuzumab was well tolerated for inoperable or recurrent breast cancer patients.

1081 Background: In patients with brain metastasis (BM) of melanoma or lung cancer, significant activity of immune checkpoint inhibitors has been reported. However, details of the immune microenvironment in BM has not been unveiled. In this study, we used immunohistochemistry (IHC) to compare primary breast tumors and BM tumor samples with respect to tumor infiltrating lymphocytes (TILs) and tumor characteristics related to the immune system. Methods: We retrospectively identified 107 patients with breast cancer, diagnosed with BM, who had undergone surgery between 2001 and 2012 at 8 institutions. We collected 191 samples which included both BM samples alone and pair-matched samples (primary and BM). Hematoxylin and eosin (H&E) stained slides were evaluated for stromal TILs in 10% increments (0–1%, > 1– < 10%, 10%–100%). IHC was performed using the following primary antibodies: CD4, CD8, Foxp3, PD-L1, PD-L2 and HLA class I. The cells positive for each antibody signal were counted automatically using ImageJ (NIH). The expression of PD-L1, PD-L2, and HLA on the tumor cells was scored as 0 (negative), 1 (weak or focal), or 2 (strong). Results: The median category of TILs of BM tumors was > 1– < 10% (range: 1–30%). Forty-six pair-matched samples were analyzed and the percentage of TILs in the primary breast tumor was significantly higher than that in BM samples (paired t-test, P < 0.01). The number of CD4/CD8/Foxp3 positive cells in primary breast tumor was also significantly higher than in BM samples (paired t-test, P < 0.05 for all categories). The negative/positive conversion occurred with the expression of HLA/PD-L2 on tumor cells (paired t-test, P = 0.03/0.06, respectively). No significant difference was observed in the overall survival (OS) of patients, from initial BM, based on high or low TILs (log-rank test, P = 0.131). However, triple negative breast cancer patients with low TILs had significantly shorter OS compared with patients with high TILs (log-rank test, P = 0.04). Conclusions: We demonstrated that TILs in BM tumors was significantly lower as compared to primary breast tumors. The expression of immune related molecules on tumor cells was converted in BM tumors.

To clarify the tolerance and pharmacokinetics of combined therapy with S-1 and trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic or recurrent breast cancer. From January 2008 through to September 2009, combined therapy with S-1 and trastuzumab was given to 7 patients with HER2-positive metastatic or recurrent breast cancer. The incidence of adverse events and the pharmacokinetics of tegafur, 5-fluorouracil, and gimeracil in plasma were studied. One patient had grade 3 leukopenia, and another had a grade 3 elevation of alanine aminotransferase. All other adverse events were grade 2 or lower. The combination of S-1 and trastuzumab did not cause any new adverse events. The incidence of adverse events was similar to those associated with S-1 alone. The median number of treatment cycles was 11. The pharmacokinetics of tegafur, 5-fluorouracil, and gimeracil after treatment with S-1 plus trastuzumab did not markedly differ from those after S-1 alone. Combined therapy with S-1 and trastuzumab did not cause any new adverse events, administration continuity was good, and the therapy was well tolerated.

Background: Our previous predictive peptide binding studies indicated that a novel 20-mer multiple antigen peptide, CH401MAP, containing an anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody epitope (N163-182), may potentially bind to more than 95% of class I human leukocyte antigens (HLAs) and to 30-50% of class II HLAs expressed on peripheral blood mononuclear cells (PBMCs). In this study, CH401MAP was used for in vitro stimulation of PBMCs obtained from Japanese breast cancer patients, and anti-CH401MAP antibody secretion was evaluated. Methods: PBMCs of breast cancer patients were stimulated with CH401MAP peptide in vitro. Eight days after stimulation, the culture supernatants were collected and the anti-CH401MAP antibody levels were determined using enzyme-linked immunosorbent assay . The correlation of the antibody level and HER2 expression level after in vitro stimulation was also evaluated. Results: CH401MAP specific antibody was detected in the culture supernatants of patients’ PBMCs after in vitro culture, irrespective of the peptide stimulation. The antibody levels of the three patient’s groups were significantly higher than that of HD group. Significant correlation was not observed between specific antibody production and cancer progression . Conclusion: The PBMC of Japanese breast cancer patients possessed the potential of anti-CH401MAP antibody secretion. The antibody secretion level was significantly higher than that of HD. It is correlated with the expression of HER2 on the cancer tissues but not with the HER2 level in the sera of patients.

Introduction Previous retrospective studies suggest that 18FDG PET/CT (PET/CT) has superior sensitivity and specificity to bone scintigraphy (BS) in detecting breast cancer bone metastases, but the difference in efficacy between these techniques has not been confirmed. Potentially, PET/CT may detect bone metastases more accurately than BS does. To test this hypothesis, this prospective study compared the diagnostic efficacy to detect bone metastases between PET/CT and BS in breast cancer patients. We also compared the response of bone metastases assessed by the PET/CT or BS with the bone metastases. Method This single-institution prospective study included consecutive patients with breast cancer diagnosed by biopsy and suspected bone metastases at the Breast Diseases Unit at Tokai University Hospital, Kanagawa, Japan between September 2011 and March 2014. Inclusion criteria were as follows: bone pain, elevated alkaline phosphatase, elevated tumor marker, and suspected bone metastases on BS. Two nuclear medicine physicians interpreted the PET/CT and BS images. Bone involvement was confirmed by biopsy, especially in the case of oligometastasis. If biopsy proved difficult to perform, conventional imaging and additional directed radiological studies and follow up were helpful. This study was approved by the Institutional Review Board of the Tokai University School of Medicine and is registered with UMIN, number 000006003. All patients provided informed consent. Result Thirty patients were initially enrolled, but two patients were excluded from analysis because they declined further follow-up imaging. The median patient age at diagnosis was 59 years (range, 31–74 years). Among the 28 patients, bone pain was observed in 6 patients, elevated alkaline phosphatase in 4, elevated tumor marker in 17, and suspected bone metastases were detected on BS in 7 patients. Among 10 patients were diagnosed bone metastases, PET/ CT detected 10 of 10 bone metastases, however BS detected 7 of 10 bone metastases. PET/CT and BS were not highly concordant in detecting osseous metastases; among 19/28 paired studies (68%), 2 (10%) were positive for metastasis, and 17 (90%) were negative. Nine occurrences (32%) were discordant; of these, 2 of 9 were PET/CT positive and BS negative; 5 of 9 were PET/CT positive and BS equivocal; one case was PET/CT negative and BS equivocal; and one was PET/CT equivocal and BS negative. Conclusion This study supports the use of PET/CT for detecting suspected osseous metastases. A large prospective study is needed to determine whether PET/CT could replace bone scintigraphy in detecting suspected bone metastases. Citation Format: Naoki Niikura, Jun Hashimoto, Toshiki Kazama, Jun Koizumi, Rin Ogiya, Mayako Terao, Risa Oshitanai, Toru Morioka, Banri Tuda, Takuho Okamura, Yuki Saito, Yutaka Imai, Yutaka Tokuda. Diagnostic performance of PET/CT and bone scintigraphy in breast cancer patients with suspected bone metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-03-01.

Immunohistochemical (IHC) expression of Ki67 has a prognostic and predictive value for breast cancer, and the IHC Ki67 labeling index is estimated by counting the number of positive and negative cells. It has not been clarified whether IHC Ki67 estimated using a semiquantitative scoring system has a prognostic value. We aimed to estimate the usefulness of scoring categories of IHC Ki67 as a prognostic factor for breast cancer subgroups.We retrospectively identified patients in the Tokai University breast cancer database for whom IHC Ki67 data were available between January 1, 2000 and December 31, 2010. Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test.Of the 1331 primary breast cancer patients included in the study, In patients with estrogen receptor (ER)-positive and HER2-negative tumors (n = 971), high and intermediate Ki67 scores were associated with poorer relapse-free survival than low Ki67 scores (P.001 and P = .002, respectively). Furthermore, in the multivariate analyses of this subgroup, progression-free survival (PFS) was significantly longer in patients with low Ki67 scores than in patients with high Ki67 scores (hazard ratio, 0.387; 95% confidence interval, 0.233-0.643; P.001). In the multivariate analyses, the Ki67 score was not significantly associated with PFS in the ER-positive and HER2-positive, ER-negative and HER2-positive, or ER-negative and HER2-negative subgroups.Our data demonstrated that low, intermediate, and high Ki67 scores have a prognostic value in breast cancer patients with ER-positive and HER2-negative tumors.

Aim: Brain metastases (BM) are associated with impaired quality of life and have an increasing problem in the management because of progressive neurological impairments. Although BM are less common in patients (pts) with breast cancer, they are associated with considerably poorer prognosis and are less responsive to systemic therapies. Recently, a trend of increased incidence of BM has been noted. To define prognostic factors for BM, compare their clinical courses and prognoses according to the subtypes, and analyze the causes of death. Methods: We retrospectively collected cohort data for 1466 pts diagnosed with BM between 2001 April and 2012 December from 24 institutions of the breast division of the Japan Clinical Oncology Group (JCOG). Results: After exclusion 210 pts' data due to the lack of enough data, overall 1256 pts with BM were evaluated. The median overall survival (OS) was 8.7 months (m) (95% CI, 7.8–9.6). Univariate and multivariate analyses revealed that pts with BM within 6 m of metastatic breast cancer diagnoses, asymptomatic brain disease, or HER2-positive/ER-positive tumors had increased OS. Median OS after BM was 9.3 m (95% CI, 7.2–11.3) for the luminal type, 16.5 m (11.9–21.1) for the luminal-HER2 type, 11.5 m (9.1–13.8) for the HER2 type, and 4.9 m (3.9–5.9) for the triple-negative type. The duration from MBC diagnosis until BM was longer in pts with luminal-type tumors than in pts with luminal-HER2 (HR = 1.24, P= 0.03), HER2 (HR = 1.83, P Conclusions: The prognosis and clinical course of pts before and after developing BM vary according to the subtype. Good prognostic factors for OS included the early detection of BM, asymptomatic brain disease, and HER2/ER-positive status. Focusing on the subtypes can optimize the prevention, early detection, and improved treatment of BM. Disclosure: All authors have declared no conflicts of interest.

In previous decades, numerous attempts have been made to develop therapeutic peptide vaccines for cancer. However, the HLA (Human Leukocyte Antigen) types are limited because most peptide vaccines are specific to the major HLA types of the area. Peptide vaccines specific for Caucasians thus may not be specific to Japanese. Moreover, they are not designed to stimulate both helper and killer T cells. We are trying to make a peptide vaccine specific to the MHC of Japanese patients that stimulates both helper and killer T cells. We selected a new-HER2 peptide including a B-cell epitope which has anti-tumor effects in a mouse system. The B-cell epitope was determined for a H401 monoclonal antibody (mAb) specific for HER2. As for epitope mapping of the chimera mAb CH401, enzyme-linked immunosorbent assay was employed with 20mer MAPs carrying a partial HER2 sequence. The CH401 epitope was determined as N:163-182, and the CH401MAP including the epitope induced anti-tumor effects in HER2-overexpressing tumor cells in a mouse system. We predicted the peptide MHC affinity and examined the in vitro reaction of PBMCs from Japanese breast cancer patients. The study enrolled 173 female breast cancer patients who underwent surgery between October 2010 and July 2012 at Tokai University Hospital. We used SYFPEITHI, BIMAS and IEDB algorithms to estimate peptide and HLA affinity. Lymphocyte proliferation ability, cell surface marker expression, cytokine (interleukin (IL)-2, IL-4 and interferon (IFN)-g) secretion and specific antibody production were analyzed in vitro. According to the algorithms, 97.1% of patients showed high to intermediate affinity of the CH401 epitope peptide to Japanese major HLA class I. Similarly, 34.5% of patients showed high to moderate affinity to HLA class II. The proliferative ability of patient groups was significantly higher than that of the HD group (HER2 0 group, p Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-11.