Banri Tsuda

Immunohistochemical Ki67 labeling index has similar proliferation predictive power to various gene signatures in breast cancer.

The objective of this study was to examine the association between the immunohistochemical Ki67 labeling index (IHC Ki67), Ki67 mRNA expression level, and first‐generation gene signatures in a cohort of breast cancer patients. We assessed associations between IHC Ki67 and first‐generation gene signatures in a panel of 39 tumor samples, using an oligonucleotide microarray. Gene expression analyses included Ki67 alone (MKi67), 21‐gene signature, mitosis kinome score signature, and genomic grade index. Correlation coefficients were calculated by Spearmans rank correlation test. In all cases, IHC Ki67, MKi67, and three genetic markers were highly correlated (ρ, 0.71–0.97). Estrogen receptor (ER)‐positive cases showed strong correlations between IHC Ki67 and other signatures (ρ, 0.79–0.83). The ER‐negative cases showed slightly lower correlations (ρ, 0.58–0.73). In ER‐positive cases, the low IHC Ki67 group showed significantly longer relapse‐free survival than the high IHC Ki67 group (P = 0.007). This difference was confirmed by multivariate analysis. Our data indicate that IHC Ki67 shows similar predictive power for proliferation in ER‐positive cancers as genomic markers. Further study of IHC Ki67 is needed to define prognostic factors and predictive factors for chemotherapy using central laboratory assessment. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02319.x, 2012)

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.

A Human B cell Receptor Epitope-Based Erbb-2 Peptide (N: 163-182)with Pan-Reactivity to the T cells of Japanese Breast Cancer Patients

Background: While monoclonal antibodies are widely accepted as being powerful in molecular targeting therapy, the activation of immunity by the B cell receptor epitope peptide has not been reported. Our objective was to evaluate a 20-mer multiple antigen peptide containing the anti-erbB-2 (HER2/neu) monoclonal antibody epitope (N: 163–182), designated as CH401MAP, as a general peptide vaccine for Japanese breast cancer patients. Methods: The 20-mer CH401 epitope peptide binding to the human leukocyte antigen (HLA) of Japanese breast cancer patients was predicted by algorithms from the following databases: SYFPEITHI, Bioinformatics and Molecular Analysis Section (BIMAS), and Immune Epitope Database (IEDB). Peripheral blood mononuclear cells (PBMCs) were collected from 173 breast cancer patients and stimulated with the peptide in vitro. Cytokine secretion was examined by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis was performed to detect CD4 and CD8 T cells and Treg cells. Results: Using the algorithms used for the simulation, CH401MAP was predicted to bind to more than 90% of class I HLAs and to 30–50% of class II HLAs in breast cancer patients. In vitro stimulation of patients’ PBMCs with CH401MAP induced a significant increase in interleukin-2 secretion, proliferation, and CD8 T cell ratios. Conclusions: Our study demonstrates that CH401MAP induces T cell activation in PBMCs derived from breast cancer patients. This antibody epitope-based peptide with HLA motifs could be a candidate peptide vaccine for general breast cancer.

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.

NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination

Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs) are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD), which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG) mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg). After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2) cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP) or keyhole limpet hemocyanin (KLH) successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

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 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.