Ayane Yamaguchi

Assessing the effects of neoadjuvant chemotherapy on lymphatic pathways to sentinel lymph nodes in cases of breast cancer: Usefulness of the indocyanine green-fluorescence method

BACKGROUNDS It is not clear how lymphatic pathways to the sentinel lymph node (SLN) change during neoadjuvant chemotherapy (NAC) for breast cancer. METHODS Using the indocyanine green (ICG)-fluorescence method, we compared lymphatic pathways to the SLN (sentinel lymphatic pathways) and SLN location before and after NAC in 36 patients (38 breasts). RESULTS Despite that 42.8% of the sentinel lymphatic pathways were changed by NAC, the locations of the SLNs were not affected by NAC. CONCLUSION These results suggest that the true SLN can be detected even after NAC, and that SLNB can be performed after NAC for clinically node-negative patients.

Indication of sentinel lymph node biopsy with breast-conserving surgery for ductal carcinoma in situ considering lymphatic flow.

192 Background: Ductal carcinoma in situ (DCIS) has been regarded as curable with resection, but axillary lymph node metastases have been reported in 2% of DCIS patients. Even when DCIS has been diagnosed by preoperative core needle biopsy (CNB), 8% to 38% of the patients have been found to have invasive ductal carcinoma (IDC) on the basis of pathological diagnosis after surgical treatment. The indication of sentinel lymph node biopsy (SLNB) and breast-conserving surgery (BCS) for DCIS is still controversial. METHODS SLNB is a standard surgical technique for early breast cancer treatment, and indocyanine green (ICG) fluorescence method is remarkable in terms of the visualization of lymphatic flow. We analyzed the variation in lymphatic drainage routes from the nipple to the SLN (sentinel lymphatic routes) by using the ICG florescence method in early breast cancer patients and investigated the effects on the localization of the tumor to the sentinel lymphatic routes after BCS. RESULTS From November 2010 to April 2012, we recorded the sentinel lymphatic routes in 118 patients. All the routes passed through the upper outer quadrant (UOQ) area, and there were more than 2 routes in 53 cases. Of these routes, 73% passed through only the UOQ area and 27% passed through the UOQ via the upper inner, lower inner, and/or lower outer quadrant area. CONCLUSIONS We should confirm the sentinel lymphatic routes by using the ICG florescence method before BCS for preoperatively diagnosed DCIS. If the lymphatic routes do not pass over the extent of resection of BCS, we can omit SLNB in the first surgical treatment and await the final pathological result. However, we should perform SLNB in addition to BCS in cases in which the lymphatic routes pass over the tumor in the region except the UOQ area.

Gene expression profile of peripheral blood mononuclear cells may contribute to the identification and immunological classification of breast cancer patients

BackgroundIt has been reported that the gene expression profile of peripheral blood mononuclear cells (PBMCs) exhibits a unique gene expression signature in several types of cancer. In this study, we aimed to explore the breast cancer patient-specific gene expression profile of PBMCs and discuss immunological insight on host antitumor immune responses.MethodsWe comprehensively analyzed the gene expression of PBMCs by RNA sequencing in the breast cancer patients as compared to that of healthy volunteers (HVs). Pathway enrichment analysis was performed on MetaCoretm to search the molecular pathways associated with the gene expression profile of PBMCs in cancer patients compared with HVs.ResultsWe found a significant unique gene expression signature, such as the Toll-like receptor (TLR) 3- and TLR4-induced Toll/interleukin-1 receptor domain-containing adapter molecule 1 (TICAM1)-specific signaling pathway in the breast cancer patients as compared to that of healthy volunteers. Distinctive immunological gene expression profiles also showed the possibility of classifying breast cancer patients into subgroups such as T-cell inhibitory and monocyte-activating groups independent of known phenotypes of breast cancer.ConclusionsThese preliminary findings suggest that evaluation of gene expression patterns of PBMCs might be both a less invasive diagnostic procedure and a useful way to reveal immunological insight of breast cancer, including biomarkers for cancer immunotherapy, such as immune checkpoint inhibitor therapy.

Abstract 3697: The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer

Triple negative breast cancer (TNBC) is still difficult to treat partly because of lacking specific target. Although 50-70% of TNBC expresses EGFR, it is less sensitive to the treatment of EGFR inhibition for TNBC as compared to the efficacy of HER2 inhibition for HER2-positive breast cancer. Several phase II study on EGFR blockade treatment has been reported, however it has not applied in a clinical setting yet. It was reported that residual tumors after treatment with EGFR-targeted antibodies showed increased HER3 abundance leading to EGFR/HER3 receptor dimerization. The signals of HER3/EGFR dimerization to PI3K/AKT/mTOR pathways are thought to be involved in cancer survival, proliferation and also up-regulation of PD-L1 expression. Thus, we hypothesized that up-regulation of HER3 signal caused by anti-cancer treatment might induce PD-L1 expression and inhibit host anti-tumor immunity. In this study, we tested the relationships between HER3 signal and PD-L1 expression by using three basal-like breast cancer cell line; MDA-MB-231, HCC70, and MDA-MB-468. MDA-MB-231 is HER3-negative, and HCC70 and MDA-MB-468 are HER3-positive cell lines. We added neureglin 1 (NRG1: HER3 ligand) to those three cell lines and analyzed PD-L1 expression of protein by flowcytometry and mRNA by qRT-PCR. Both protein and mRNA level of PD-L1 on HCC70 and MDA-MB-468 treated with NRG1 are increased as compared with those without NRG1 while there was no change of PD-L1 expression of MDA-MB-231 either with or without NRG1. In order to confirm the significance of potential treatment target of HER3, we evaluated HER3 expression in biopsy samples by immunohistochemistry before neoadjuvant chemotherapy (NAC) including all phenotypes. Thirteen pathological complete response (pCR) cases after NAC and 6 non pCR cases were included. We scored the HER3 stainability from 0 to 3 and found that non pCR cases showed significantly higher HER3 score than pCR cases (84.6% and 33% respectively, p=0.0149). Although further study is needed, these results suggest that HER3 signal possibly regulates PD-L1 expression in HER3-positive basal-like breast cancer and treatment with anti-HER3 targeting therapy combination with an immune checkpoint inhibition therapy for HER3 positive NAC resistant patients might be warranted. Note: This abstract was not presented at the meeting. Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Masakazu Toi. The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3697. doi:10.1158/1538-7445.AM2017-3697

Abstract PD4-08: Efficacy of compression therapy using surgical gloves for nanoparticle albumin-bound-paclitaxel-induced peripheral neuropathy: A phase II multicenter study by the Kamigata breast cancer study group

PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) is an adverse effect of many commonly used chemotherapeutic agents, including taxanes. However, there is currently no established effective prophylactic management for CIPN. Thus, we investigated the efficacy of using surgical glove (SG) compression therapy to prevent nanoparticle albumin-bound-paclitaxel (nab-PTX)-induced peripheral neuropathy. PATIENTS AND METHODS: Patients with primary and recurrent breast cancer who received 260 mg/m2 of nab-PTX were eligible for this case-control study. The patients wore two SGs of the same size, that is, one size smaller than the size that fit, on their dominant hand for 90 minutes. They did not wear SGs on the non-dominant hand, which served as the control hand. Peripheral neuropathy was evaluated at each treatment cycle using Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 and the Patient Neurotoxicity Questionnaire (PNQ). The temperatures of each fingertip of the compression SG-protected and control hands were measured by using thermography. RESULTS: Between August 2013 and January 2016, 43 patients were enrolled, and 42 were evaluated. As shown in Table 1, the overall occurrence of ≥grade 2 sensory and motor peripheral neuropathy according to the CTCAE was significantly lower in the SG-protected hands than in the control hands (76.1% vs. 21.4% and 57.1% vs. 26.2%, respectively, p No patients withdrew from this study because they could not tolerate the compression from the SGs. The mean temperature of each fingertip significantly decreased (1.42–2.60 °C) in the SG-protected hands compared to in the control hands. CONCLUSIONS: SG compression therapy appears effective for reducing nab-PTX-induced peripheral neuropathy. The nab-PTX exposure to the peripheral nerve may be decreased because the SG decreases microvascular flow to the fingertip. Citation Format: Tsuyuki S, Senda N, Kanng Y, Yamaguchi A, Yoshibayashi H, Kikawa Y, Katakami N, Kato H, Hashimoto T, Okuno T, Yamauchi A, Inamoto T. Efficacy of compression therapy using surgical gloves for nanoparticle albumin-bound-paclitaxel-induced peripheral neuropathy: A phase II multicenter study by the Kamigata breast cancer study group [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 PD4-08.

Abstract 5706: Gene expression profile of peripheral blood mononuclear cells in breast cancer patients may be contribute to the identification and the immunological classification of breast cancer patients by blood test

It is reported that gene expression profile of peripheral blood mononuclear cells (PBMCs) exhibits unique gene expression signature in cancer patients including renal cell carcinoma, pancreatic cancer and lung cancer. Since pancreatic cancer diagnosis is difficult in not only early detection of the disease but also in the diagnosis itself, development of novel diagnostic tools in addition to conventional diagnostic strategy has been awaited. On the other hand, in breast cancer, because early diagnosis by mammography and ultra sound examination on breast is established successfully, exploration of gene expression profile of PBMCs may be important in terms of insight to host antitumor immune response aspects. In the current study, we performed RNA sequencing (RNA-seq) analysis on RNA of PBMCs from 3 healthy volunteers, 6 early and 7 metastatic breast cancer patients including all phenotypes defined by ER, PgR and HER2. Genes that showed FDR These findings suggested that evaluation of gene expression patterns of PBMCs of breast cancer patients might distinguish breast cancer patients from healthy subjects and the gene expression signature of PBMCs which divided breast cancer patients into 3 groups might reveal immunologically important biologic properties such as response prediction of cancer immunotherapy including immune checkpoint inhibition treatment. Citation Format: Eiji Suzuki, Kosuke Kawaguchi, Masahiro Sugimoto, Fengling Pu, Ryuji Uozumi, Ayane Yamaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Satoshi Morita, Masakazu Toi. Gene expression profile of peripheral blood mononuclear cells in breast cancer patients may be contribute to the identification and the immunological classification of breast cancer patients by blood test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5706. doi:10.1158/1538-7445.AM2017-5706

Downregulation of neuropilin-1 on macrophages modulates antibody-mediated tumoricidal activity

Neuropilin-1 (NRP-1)-expressing macrophages are engaged in antitumor immune functions via various mechanisms. In this study, we investigated the role of NRP-1 on macrophages in antibody-mediated tumoricidal activity. Treatment of macrophages with NRP-1 knockdown or an anti-NRP-1-neutralizing antibody significantly suppressed antibody-dependent cellular cytotoxicity and modulated cytokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model bearing human epidermal growth factor receptor-2 (HER2)-positive breast cancer xenografts showed that antibody-mediated antitumor activity and tumor infiltration of CD4+ T lymphocytes were significantly downregulated when peripheral blood mononuclear cells in which NRP-1 was knocked down were co-administered with an anti-HER2 antibody. These results revealed that NRP-1 expressed on macrophages plays an important role in antibody-mediated antitumor immunity. Taken together, the induction of NRP-1 on macrophages may be a therapeutic indicator for antibody treatments that exert antibody-dependent cellular cytotoxicity activity, although further studies are needed in order to support this hypothesis.

Abstract 4142: Proteomics analysis of breast cancer cell-specific proteins that are transferred to immune cells via trogocytosis

We previously reported that HER2 is transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis (Suzuki et al. BMC cancer 2015) and EGFR of MDA-MB-231 (triple negative human breast cancer cell line) is also transferred to immune cells via trogocytosis and those immune cells express EGFR on the cell surface without therapeutic antibody administration (AACR 2015 #2349). There are several reports that suggest that trogocytosis might act to stimulate immunological tolerance or immune effector cell activation, however its immunological roles still remain unclear. Therefore, in order to clarify the role of trogocytosis, we study the profile of proteins that potentially are transferred from cancer cell to immune cell via trogocytosis. Human triple negative breast cancer cell line, MDA-MB-231 that was cultured with the medium containing arginine labeled with 13C instead of normal 12C (SILAC method, ThermoFisher Scientific) was co-cultured with human peripheral blood mononuclear cell (PBMC) that was cultured with normal medium. The cell mixture was labeled with CD45+ magnetic beads and CD45+ cell (PBMC) and CD45- cell (MDA-MB-231) were separated by MACS cell separation kit (Miltenyi Biotec). Proteins of PBMC that were passed the trogocytosis were resolved on SDS-PAGE and LC-MS/MS was carried out to identify the cancer specific trogocytosed proteins in PBMC by SILAC method. We found that proteins with higher H/L ratio were included in many of cytoskeletal proteins and interestingly mitochondrial metabolism related proteins of cancer cells were also detected in PBMC. Those up-regulated proteins in PBMC were reduced in MDA-MB-231. The findings suggested that the role of trogocytosis of PBMC on breast cancer cell might be involved in metabolisms of both cancer cell and immune cell. Thus, we are exploring the effect of trogocytosis on cancer cell metabolisms especially on mitochondrial related metabolism that could be reduced by immune cell trogocytosis and also on metabolism of immune cells themselves that capture the cancer cell proteins via trogocytosis. Citation Format: Eiji Suzuki, Masashi Inoue, Shinji Ito, Junko Satoh, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Masakazu Toi. Proteomics analysis of breast cancer cell-specific proteins that are transferred to immune cells via trogocytosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4142.

Abstract 4154: Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer

Tumor-infiltrating immune cells (TIIs) in HER2+ breast cancer (BC) play an important role in treatment with anti-HER2 therapy. However, the precise mechanisms of how TIIs exert anti-tumor activity remain unclear. Neuropilin-1 (NRP-1) on macrophages regulates immune functions in various cancers and thus we explored the role of NRP-1 expressing macrophages in anti-tumor activity in HER2+ BC. We show that NRP-1 on macrophages regulated the migration of and chemokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model showed that NRP-1 knockdown of macrophages in adoptively transferring peripheral blood mononuclear cells (PBMCs) suppressed anti-tumor activity and infiltration of CD45+ immune cells into tumors. Interestingly, NRP-1 expressing TIIs were mainly CD4+ T cells, despite little expression of NRP-1 on CD4+ T cells in PBMCs. We found that NRP-1 expression on CD4+ T cells was induced by NRP-1 transfer from macrophages to T cells. In HER2+ BC patients, NRP-1 expressing TIIs correlated with better clinical outcomes. These results demonstrate that NRP-1 expressing macrophages are key subsets of immune cells in trastuzumab-mediated anti-tumor activity and may predict better outcomes for HER2+ BC patients. In conclusion, NRP-1 expression is required for differentiation and activation of macrophages. NRP-1 expressing macrophages promote NRP-1 expression on CD4+ T cells by direct interaction and contribute to anti-tumor immune responses in HER2+ BC. Citation Format: Kosuke Kawaguchi, Eiji SUzuki, Masashi Inoue, Isao Kii, Tatsuki R. Karaoke, Masahiro Hiram, Keiko Iwaisako, Pu Fengling, Mariko Niche, Ayane Yamaguchi, Hironori Haga, Masatoshi Hagiwara, Masakazu Toi. Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4154.

Abstract 4149: Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1

BACKGROUND: We previously reported that direct co-culture of triple negative breast cancer cell line MDA-MB-231 and immune cells results in reduction of EGFR expression on cell surface of MDA-MB-231 (AACR 2015 #2349). However, a role of reduction of EGFR on MDA-MB-231 via co-culture with immune cells still remains unclear. Therefore, we evaluated a role of reduction of EGFR on MDA-MB-231 at the immunological point of view by testing expression of immune related genes including PD-L1. METHODS: In order to verify the importance of direct immune cell contact to breast cancer cell, MDA-MB-231 cells were co-cultured directly or indirectly with THP-1 cells (human monocytic cell line) at 1:50 cellular ratios. In order to study indirect co-culture assay, we used cell culture insert to avoid direct cancer cell-immune cell contact. We analyzed gene expression by quantitative real time PCR and membrane protein expression by flow cytometry of EGFR, also other HER family on MDA-MB-231 which is directly or indirectly co-cultured with THP-1. We also evaluated the expression of immune related genes including PD-L1. RESULTS: Both mRNA and protein level of EGFR on MDA-MB-231 cells directly co-cultured with THP-1 were significantly decreased as compared to those with indirectly co-cultured MDA-MB-231 cells. There are no significant differences in EGFR expression between indirectly co-cultured MDA-MB-231 cells and control MDA-MB-231 cells. Importantly, PD-L1 expression on MDA-MB-231 cells directly co-cultured with THP-1 was significantly decreased as compared to that with indirectly co-cultured MDA-MB-231 cells. CONCLUSION: It has been reported that PD-L1 expression in cancers is regulated by phosphatidylinositol 3-kinase (PI3K) and Akt signaling. Thus, our findings may give a novel insight on regulation of PD-L1 expression on cancer cells in tumor microenvironment that tumor infiltrated immune cell directly contact with cancer cells and EGFR down-regulation leads to reduction of PD-L1 expression on cancer cells. Further investigation is needed to elucidate the mechanism of reduction of EGFR by direct immune cell contact to cancer cells and its interaction with modulation of PD-L1 expression. This will provide novel aspects for immune therapy of breast cancer patients. Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Masakazu Toi. Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4149.