Mayako Terao

Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

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.

Prognostic Significance of the Ki67 Scoring Categories in Breast Cancer Subgroups

BACKGROUND 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. PATIENTS AND METHODS 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. RESULTS 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. CONCLUSION 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.

The Effect of Peptide Treatment on the HLA-Binding and Antibody Production in Peripheral Blood Mononuclear Cells Obtained from Japanese Breast Cancer Patients

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.

Post-marketing Safety Evaluation of S-1 in Patients with Inoperable or Recurrent Breast Cancer: Especially in Patients Treated with S-1 + Trastuzumab

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 + trastuzumab. Conclusions Monotherapy with S-1 or combination therapy with S-1 + trastuzumab was well tolerated for inoperable or recurrent breast cancer patients.

Feasibility and pharmacokinetics of combined therapy with S-1 and trastuzumab in patients with human epidermal growth factor receptor 2-positive metastatic or recurrent breast cancer

BackgroundTo 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.MethodsFrom 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.ResultsOne 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.ConclusionsCombined therapy with S-1 and trastuzumab did not cause any new adverse events, administration continuity was good, and the therapy was well tolerated.

Comparison of Ki-67 labeling index measurements using digital image analysis and scoring by pathologists

BackgroundRoutine 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.MethodsWe 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.ResultsBased 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 < 0.001) and obtained using image analysis (p = 0.031).ConclusionsThe Ki-67 indices obtained using image analysis were moderately correlated with those scored by pathologists. Digital image analysis can be effective for measuring Ki-67 values, because they are associated with relapse-free survival in estrogen receptor-positive, human epidermal growth factor receptor 2-negative, and patients at stage I or II.

Abstract P2-04-13: Difference of immune microenvironment between primary and recurrent tumours in breast cancer patients

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 ( 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.

Abstract P3-03-01: Diagnostic performance of PET/CT and bone scintigraphy in breast cancer patients with suspected bone metastasis

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.

Abstract P5-01-11: A new anti-HER2 peptide “CH401MAP” can stimulate the immunity of breast cancer patients

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.

Abstract P3-13-02: Safety and Efficacy of Zoledronic Acid Beyond 24 Months in Breast Cancer Patients

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.