Mariko Nishie

Trogocytosis-mediated expression of HER2 on immune cells may be associated with a pathological complete response to trastuzumab-based primary systemic therapy in HER2-overexpressing breast cancer patients

BackgroundTrogocytosis is defined as the transfer of cell-surface membrane proteins and membrane patches from one cell to another through contact. It is reported that human epidermal growth factor receptor 2 (HER2) could be transferred from cancer cells to monocytes via trogocytosis; however, the clinical significance of this is unknown. The aim of this study is to demonstrate the presence and evaluate the clinical significance of HER2+ tumor-infiltrated immune cells (arising through HER2 trogocytosis) in HER2-overexpressing (HER2+) breast cancer patients receiving trastuzumab-based primary systemic therapy (PST).MethodsTo assess the trogocytosis of HER2 from cancer cells to immune cells, and to evaluate the up- and down-regulation of HER2 on immune and cancer cells, peripheral blood mononuclear cells from healthy volunteers and breast cancer patients were co-cultured with HER2+ and HER2-negative breast cancer cell lines with and without trastuzumab, respectively. The correlation between HER2 expression on tumor-infiltrated immune cells and a pathological complete response (pCR) in HER2+ breast cancer patients treated with trastuzumab-based PST was analyzed.ResultsHER2 was transferred from HER2+ breast cancer cells to monocytes and natural killer cells by trogocytosis. Trastuzumab-mediated trogocytosed-HER2+ effector cells exhibited greater CD107a expression than non-HER2-trogocytosed effector cells. In breast cancer patients, HER2 expression on tumor-infiltrated immune cells in treatment naïve HER2+ tumors was associated with a pCR to trastuzumab-based PST.ConclusionsHER2-trogocytosis is visible evidence of tumor microenvironment interaction between cancer cells and immune cells. Given that effective contact between these cells is critical for immune destruction of target cancer cells, this interaction is of great significance. It is possible that HER2 trogocytosis could be used as a predictive biomarker for trastuzumab-based PST efficacy in HER2+ breast cancer patients.

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 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 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.

Abstract 720: Downregulation of ATP6V1B1 in HER2-overexpressing human breast cancer cell line leads resistance against trastuzumab-mediated antibody-dependent cellular cytotoxicity

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: PIK3CA mutation has been becoming widely recognized as a resistant mechanism or therapeutic biomarker for trastuzumab based anti HER2 targeting therapy, however little is clear on resistant mechanism of trastuzumab mediated antibody dependent cellular cytotoxicity (ADCC) in HER2 overexpressing (HER2+) breast cancer patients. In the current study, we investigated the role of Vacuolar-ATPase subunit ATP6V1B1 on the resistant mechanism of trastuzumab mediated ADCC in HER2+ human breast cancer cell line SK-BR-3. Methods and Results:To establish the trastuzumab mediated ADCC resistant SK-BR-3, isolated healthy PBMCs are co-cultured with 1-10 μg/ml of trastuzumab and SK-BR-3 for 4-9 hours. The cell mixture was incubated with CD45 magnetic beads and CD45 positive PBMCs were removed from the mixture using MACS cell separation system (Miltenyi Biotec, Germany). The survived SK-BR-3 thereafter co-cultured with 1-10 μg/ml of trastuzumab and PBMCs for several times. After 7 passages of the resistant cell making procedure (approximately for 6 months), cytotoxicity was measured by conventional ADCC assay (E/T ratio = 10/1) with various concentration of trastuzumab. The cytotoxicity of ADCC resistant SK-BR-3 was significantly reduced as compared with normal SK-BR-3 cytotoxicity in 10μg/ml of trastuzumab (15.9% and 42.8%, respectively). Then, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray to sort candidate genes for the resistance. Several genes were up- or down-regulated in ADCC resistant SK-BR-3 and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. To clarify the result of comprehensive gene expression analysis, we investigated ATP6V1B1 mRNA level of SK-BR-3 and resistant cell by using real time PCR and found the 50% reduction of ATP6V1B1 mRNA level in ADCC resistant cells. Thus, to evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, ATP6V1B1-knocked down SK-BR-3 (sh-RNA knockdown method) were tested in trastuzumab mediated ADCC assay and found that ATP6V1B1-knocked down SK-BR-3 were significantly less ADCC activity with LDH assay as compared to control SK-BR-3 (19.1% and 40.0%, respectively).Conclusions: ATP6V1B1 might be one factor for trastuzumab mediated ADCC resistance in HER2+ breast cancer cells. Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Keiko Sakamoto, Yuji Fukushima, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Downregulation of ATP6V1B1 in HER2-overexpressing human breast cancer cell line leads resistance against trastuzumab-mediated antibody-dependent cellular cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2015-720

Abstract 1272: Upregulation of mRNA of CD80, FOXP3, NKG2D and PD-L1 in peripheral blood mononuclear cells is associated with metastatic breast cancer patients:

BACKGROUND: Immune cells in the blood circulation may directly or indirectly interact with cancer cells including circulating tumor cells (CTCs), local and metastatic lesions during immune surveillance. Upon such interactions, the immune cells might be activated or inhibited in anti-rumor responses with several immune checkpoint pathways. However, little is known about alteration of immune checkpoint pathways in peripheral blood mononuclear cells (PBMCs) in the process of breast cancer progression. In the current study, we investigated the alteration of mRNA expression which is associated to immune checkpoint pathways in PBMCs of early and late stage breast cancer patients by comparing with healthy populations. PATIENTS AND METHODS: Twelve early operable breast cancer (EBC) patients, 30 metastatic breast cancer (MBC) patients in Kyoto University Hospital and 6 healthy volunteers (HV) participated. PBMCs were isolated by using cell preparation tubes according to the manufacturer9s instruction. RNA was extracted from the total PBMCs and relative mRNA expressions of CD4, CD8, CD40, CD56, CD80. CTLA4, FOXP3, IDO1, IDO2, NKG2D, NRP1, PD-1, and PD-L1 were evaluated by quantitative RT-PCR. RESULT: The expressions of mRNA were not significantly changed between HV and EBC. By contrast, between HV or EBC and MBC, significant differences were detected in mRNA expression in CD80, FOXP3, NKG2D and PD-L1. All of them were upregulated in MBC, compared with HV or/and EBC (Reference Table 1). CONCLUSION: Based on the findings of the correlation between up-regulation of the immune check point molecules tested and metastatic cancer samples, it suggested that the development of breast cancer burden should be relevant to some specific immune resistance mechanism. Further careful evaluations of the current study will provide novel prospects for diagnostic biomarkers and therapeutic targets for management of breast cancer patients. Citation Format: Ayane Yamaguchi, Kosuke Kawaguchi, Eiji Suzuki, Mariko Nishie, Masakazu Toi. Upregulation of mRNA of CD80, FOXP3, NKG2D and PD-L1 in peripheral blood mononuclear cells is associated with metastatic breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1272. doi:10.1158/1538-7445.AM2015-1272

Abstract 1092: Trogocytosis of HER2 overexpressing human breast cancer cell lines may induce its dormancy without depending on HER family signal

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We and others showed that HER2 loss of the target cancer cell was caused by shaving of HER2 by CD14+ cells (known as trogocytosis) when trastuzumab mediated antibody dependent cellular cytotoxicity (ADCC) was introduced in the HER2 positive breast cancer cells. In the current study, we report that loss of HER2 and HER3 of SK-BR-3 and BT-474 (both HER2 overexpressing human breast cancer cell lines) is caused via uptake of the receptors by CD14+ cells without trastuzumab administration. This cell-cell contact phenomenon is quite similar to the situation of the interaction between circulating tumor cell (CTC) and immune cells in the blood stream. Interestingly, the cancer cells in contact with immune cells (trogocytosis occurs) exhibited a long term dormancy in vitro. Therefore this in vitro trogocytosis concept may explain how cancer cell changes their proliferation ability into dormancy in tumor microenvironment interacting with immune cells. Briefly, SK-BR-3 or BT-474 cells were co-cultured with PBMCs for 60 to 90-min with various Effector immune cell / Target cancer cell (E/T) ratio as trogocytosis assay. The cell suspension of 90-min incubation was applied to flow cytometry to evaluate the expression level of HER1, HER2, and HER3. The cell suspension of 60-min incubation was applied to MACS cell separation kit (Miltenyi Biotec) and separated to cancer cells and immune cells. Gene expression profile of the separated cancer cells was tested by DNA microarray and the candidate up-regulated and down-regulated shedding of HER family related genes were re-confirmed by qRT-PCR thereafter. The separated cancer cells and intact SK-BR-3 were cultured and compared their proliferation ability. Higher E/T ratio induced higher loss of HER2 and HER3 on both SK-BR-3 and BT-474 while certain amount of HER2 and HER3 were still expressed on the cancer cells with an optimized E/T ratio. There was no HER1 loss on the cancer cells after trogocytosis. SK-BR-3 that underwent trogocytosis (SK-T) showed extremely low proliferation ability as compared to the intact cancer cells. The candidate genes that might be related to proteolytic shedding of HER family tested by DNA microarray were ADAM-10, -15, and -17 and MMP-1, -3, and -28. However, such different expression profile was not confirmed when the cells were tested by qRT-PCR. These findings suggested that loss of HER2 and HER3 of the cancer cells during contact with immune cells might not be affected by proteolytic activity change of ADAM or MMP. Although it is reported that the target cells after trogocytosis survive without apoptosis, our study revealed that trogocytosis with higher E/T ratio induced incomplete apoptosis of target cancer cell, which result in the cancer cell dormancy. Because the cancer cells that underwent trogocytosis became quiescent even if some extent of the receptors were present, factors other than HER family that affect on the dormancy still remain to be identified. Citation Format: Junichi Aratake, Eiji Suzuki, Natsue Uehiro, Mariko Nishie, Kosuke Kawaguchi, Fumiaki Sato, Masakazu Toi. Trogocytosis of HER2 overexpressing human breast cancer cell lines may induce its dormancy without depending on HER family signal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1092. doi:10.1158/1538-7445.AM2014-1092

Abstract 1077: Loss of HER2 via trogocytosis by CD14+ cells in HER2-overexpressing breast cancer cells is associated with diminishing of trastuzumab-mediated antibody-dependent cellular cytotoxicity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The transfer of cell-surface membrane proteins and membrane patches from one cell to another at the time of contact, known as trogocytosis, was reported in chronic lymphocytic leukemia and multiple myeloma patients and was associated with clinical outcome. However, in solid malignant tumors, such as breast cancer, there is no evidence that trogocytosis occurs clinically thus its clinical importance has not been evaluated yet. In the current study, we found significantly high HER2 expression on tumor-infiltrated CD14+ cells as compared to control CD14+ cells of PBMCs in HER2 overexpressing breast cancer patient who had been treated with trastuzmab. Digested tumor cell suspension and its autologous PBMCs were co-cultured at 1:10 ratio with 0 and 1 μg/ml of trastuzumab as trogocytosis assay. Percentage of HER2 positive CD14+ cells was higher in the treatment of 1 μg/ml of trastuzumab as compared to that of 0 μg/ml of trastuzumab (17% and 12%, respectively) while HER2 expression of digested tumor cells was lower in 1 μg/ml of trastuzumab treatment as compared to that of 0 μg/ml of trastuzumab (18% and 25%, respectively). These findings suggested that HER2 might be transferred from HER2 overexpressing breast tumor cells to CD14+ cells, which was possibility of evidence of trogocytosis in the HER2+ breast cancer patient, and suggested that CD14+ cell might have an important role in the trogocytosis. We next define the role of CD14+ cells and CD56+ cells in the process of both trogocytosis and antibody dependent cellular cytotoxicity (ADCC) in experimental setting using SK-BR-3 and BT-474, both HER2 overexpressing human breast cancer cell lines, and MCF-7 and MDA-MB-231, both HER2 negative human breast cancer cell lines. We found that trogocytosis was observed specifically on HER2 overexpressing breast cancer cell lines in in vitro torogocytosis assay and CD14+ cells have a role for trogocytosis dominantly as compared to CD56+ cells. HER2 loss was increased as Effector / Target ratio or concentration of trastuzumab was increased. HER2 loss on target cancer cells caused by trogocytosis of CD14+ cells inhibits CD56+ cell-mediated ADCC, however when CD14+ cells are depleted, such inhibitory effects are recovered and CD56+ cells show better cytotoxicity. Currently analyzed preliminary clinical sample data and experimental data of HER2 loss via trogocytosis might explain different level of treatment response of trastuzumab in individual patients. Citation Format: Eiji Suzuki, Mariko Nishie, Kosuke Kawaguchi, Sunao Tanaka, Masakazu Toi. Loss of HER2 via trogocytosis by CD14+ cells in HER2-overexpressing breast cancer cells is associated with diminishing of trastuzumab-mediated antibody-dependent cellular cytotoxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1077. doi:10.1158/1538-7445.AM2014-1077