Search

Cataract and retinopathy remain the preventable cause of blindness worldwide, and many pharmacological strategies have been proposed for the treatment of these eye diseases. Animal models play an important role in understanding the pathophysiological features of eye disease and developing for a new therapy. In this study, we investigated the development of cataract and retinal lesion with diabetes using an obese type 2 diabetic models SDT fatty rat. Macroscopic analysis in eyes was performed from 16 to 24 weeks of age and histological analysis was performed at 24 weeks of age. As a result, the lens cloudiness was observed from 19 weeks of age and the degree of the cloudiness was more progressed until 24 weeks of age. Histopathological findings, such as degeneration of lens fiber and shortening and irregular arrangement of cone and rod in retinal tissue, were observed at 24 weeks of age. In conclusion, SDT fatty rats may be useful to understand the pathological features in diabetic cataract and retinopathy develop a new therapy for the disease.

Among several mechanisms for the resistance of human epidermal growth factor receptor 2-overexpressing (HER2 +) cancer cells to trastuzumab, little is known regarding the mechanism underlying the resistance to trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Cell death due to ADCC is caused by apoptosis of target cells induced by granzymes released from natural killer cells. Because optimal granzyme physiological activity occurs at neutral pH, we assumed that the pH of the intracellular environment influences the cytotoxic effects of granzymes. We established ADCC-resistant cells and compared them with wild-type cells in terms of the expression of intracellular pH-regulating genes. The expression of ATP6V1B1, which encodes a component of vacuolar ATPases, was downregulated in the ADCC-resistant cells. Thus, to functionally characterize ATP6V1B1, we used a CRISPR/Cas9 system to generate ATP6V1B1-knockout SKBR3 and JIMT-1 cells (both HER2 + human breast cancer cell line). The resulting cells exhibited significantly less ADCC than the control SKBR3 and JIMT-1 cells. The intracellular pH of the ATP6V1B1-knockout SKBR3 and JIMT-1 cells was significantly lower than control SKBR3 and JIMT-1cells. An analysis of granzyme dynamics during the ADCC reaction in cancer cells revealed that granzymes degraded intracellularly in the control SKBR3 and JIMT-1 cells and accumulated in ATP6V1B1-knockout cells, but were not cytotoxic. These findings suggest that decreased vacuolar ATPase activity alters the cytoplasmic pH of cancer cells to create an environment that is less suitable for granzyme bioactivity, which adversely affects the induction of apoptosis of cancer cells by NK cells.

PURPOSE: To describe the foveal characteristics of children with a history of retinopathy of prematurity (ROP) using optical coherence tomography angiography (OCTA). METHODS: All eyes were examined by OCTA (RTVue AVANTI, Optovue Inc, Fremont, CA) with a scan of 3 × 3 mm cantered on the fovea. The size of the foveal avascular zone (FAZ), central retinal thickness (CRT), and foveal bulge were measured. RESULTS: Forty-eight eyes of 26 children with a history of ROP and a mean age of 8.8 years with a range of 4–16 years (ROP group) were studied. Sixty-six eyes of 36 children without any fundus abnormalities and with an average age of 10.5 years and a range of 3–17 years (control group) were studied as controls. The mean FAZ area in the ROP group was 0.18 mm(2) which was significantly smaller than the 0.32 mm(2) in the control group (p

This study aimed to evaluate the predictions of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for prognosis of triple-negative breast cancer (TNBC), especially with residual disease (RD) after preoperative chemotherapy. This retrospective analysis included 74 TNBC patients who received preoperative chemotherapy. DCE-MRI findings from three timepoints were examined: at diagnosis (MRIpre), at midpoint (MRImid) and after chemotherapy (MRIpost). These findings included cancer lesion size, washout index (WI) as a kinetic parameter using the difference in signal intensity between early and delayed phases, and time-signal intensity curve types. Distant disease-free survival was analysed using the log-rank test to compare RD group with and without a fast-washout curve. The diagnostic performance of DCE-MRI findings, including positive predictive value (PPV) for pathological responses, was also calculated. RD without fast washout curve was a significantly better prognostic factor, both at MRImid and MRIpost (hazard ratio = 0.092, 0.098, p mid was 76.7% by the cut-off point at negative WI value or lesion size = 0, and 66.7% at lesion size = 0. WI and curve types derived from DCE-MRI at the midpoint of preoperative chemotherapy can help not only assess tumour response but also predict prognosis.

It 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. We 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. We 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. These 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.

PGAM5 is a unique type of protein phosphatase that exists in mitochondria. It has been shown to exist in the inner mitochondrial membrane through its transmembrane domain and to be cleaved within the transmembrane domain upon mitochondrial dysfunction. However, its submitochondrial localization remains controversial; many researchers claim that PGAM5 localizes to the outer mitochondrial membrane based on the findings that PGAM5 associates with many cytoplasmic proteins. Here, we found that cleaved PGAM5 was released from mitochondria during mitophagy, a selective form of autophagy specific for mitochondria,and that the release was inhibited by proteasome inhibitors in HeLa cells stably expressing the E3 ubiquitin ligase Parkin.However, treatment of parental HeLa cells lacking Parkin with mitophagy-inducing agents caused PGAM5 cleavage but did not cause its release from mitochondria. Thus, cleaved PGAM5 appears to be released from mitochondria depending on proteasome-mediated rupture of the outer membrane during mitophagy, which has been previously shown to precede autophagy-mediated degradation of whole mitochondria. This study suggests that PGAM5 senses mitochondrial dysfunction in the inner mitochondrial membrane and serves as a signalling intermediate that regulates the cellular response to mitochondrial stress upon its cleavage and release from mitochondria., Journal of biochemistry, 165(1), pp.19-25; 2019

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.

PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.

Background The purpose of this study was to clarify the alterations of major immune regulators in peripheral blood mononuclear cells (PBMCs) of cancer patients and to analyze the association with the disease progression in breast cancer patients. Methods The study included 6 healthy volunteers (HVs), 12 primary breast cancer (PBC) patients, and 30 metastatic breast cancer (MBC) patients. The expression of immune regulators such as, CCR6, CD4, CD8, CD14, CD40, CD56, CD80, CTLA4, CXCR4, FOXP3, IDO-1, IDO-2, NKG2D, NRP-1, PD-1, and PD-L1 mRNA in PBMCs was measured by quantitative RT-PCR. Analysis of variance with contrasts was performed to find expression patterns of the three groups (HVs, PBC, MBC). Results We clarified the alterations of mRNA of major immune regulators PD-L1, FOXP3, CD80, CD40, and CD14 in PBMCs of cancer patients and the association of these alternations with disease progression. Furthermore, PD-L1 expression was correlated with serum interferon-γ production. Conclusion Our data suggested that mRNA expressions of PD-L1, FOXP3, CD80, CD40 and CD14 in PBMCs are affected by disease progression. Understanding the roles of these various interactions will be of importance to future studies aiming to uncover biomarkers for predicting response to immune therapy.

e12631 Background: Residual disease after preoperative chemotherapy is known to be a poor prognostic factor in triple-negative breast cancer (TNBC). Perioperative use of cisplatin (CDDP) is a well-known, standard treatment for most tumors except breast cancer. In this study, we explore the long-lasting anti-tumor activity of CDDP for TNBC patients. Methods: This research was based on consecutive study of seventy-nine previously untreated stage I–III TNBC patients treated with or without CDDP-based preoperative chemotherapy from 2007 to 2018 at the Kyoto University Hospital. We calculated the residual cancer burden (RCB) using the RCB calculator. All patients were sorted according to their RCB class: the pathological complete response (pCR), and RCB class I (minimal), II (moderate) or III (extensive). Distant disease-free survival (DDFS) was analyzed using the Kaplan-Meier method. Using mass spectrometry, we measured platinum concentrations in serum and normal breast tissue removed during surgery from patients treated with any platinum agents. We examined multiplex cytokine assays from serum sequentially, comparing CDDP and non-CDDP groups. Results: Forty-seven cases were treated with CDDP, and thirty-two did not have CDDP. The pCR rate was 53.2% for the CDDP group and 21.9% without CDDP. Patients with pCR and RCB class I were all distant disease-free. There was no significant difference between each RCB class using CDDP. However, patients belonging to RCB classes II and III with CDDP showed a better prognosis than the same RCB classes without CDDP (hazard ratio = 0.12). Platinum concentrations in serum were correlated with the time from the last dose of platinum agents, but platinum was even detected in serum two years after CDDP. No significant difference was found in the platinum concentrations of serum or normal breast tissue in the pCR and non-pCR groups. We also found that there were significant cytokine expression alteration related to T-cell infiltration only in the CDDP group one year after operation. It is suggested that CDDP may even affect the systemic immune alteration one year after operation. Conclusions: Our data shows that CDDP- based chemotherapy improves DDFS in patients with a substantial residual tumor. CDDP seems to sustain anti-tumor activity for a number of years and interaction with the systemic immune alteration.

Backgrounds The prognostic value of biomarkers in metastatic colorectal cancer (mCRC) patients with liver metastases remains unclear. We assessed the difference of expression of biomarkers between primary tumors and liver metastases treated with chemotherapy in mCRC patients, as well as the prognostic value of these markers. Methods Forty-three mCRC patients with liver-limited disease from January 2007–November 2011 were analyzed. They all received resection of primary tumors followed by oxaliplatin-based chemotherapy. After chemotherapy, they all received hepatic resection. Forty-three paired primary and metastatic tumor specimens were collected to measure the messenger RNA expression of six biomarkers by the Danenberg tumor profile method (thymidylate synthase, dihydropyrimidine dehydrogenase [DPD], excision repair cross-complementing gene1, thymidine phosphorylase [TP], folylpolyglutamate synthase, and regenerating islet-derived family, member 4). Results Thirty-six patients' messenger RNA was used for analysis. All markers showed similar expression between primary and metastatic sites. The low-expression group of Danenberg tumor profile and TP in the primary tumor showed significantly higher overall survival than the high-expression group ( P P = 0.033), but for DPD and TP in liver metastases, there were no significant differences of overall survival between the two groups. The ratios of marker expression in liver metastatic site to that in primary site of DPD and TP were significantly higher in chemo-responders than in non-chemo-responders ( P = 0.034 and P = 0.022). Conclusions Biomarkers' expressions in liver metastases were similar to those in the primary tumor. DPD and TP in the primary lesion may be a prognostic factor in chemotherapy-naive mCRC patients with liver-limited disease, but those in liver tumor were not. Further validated analysis to our results would be warranted.

The aim of this study was to examine the morphological temporomandibular joint (TMJ) changes that occur after orthodontic treatment in patients with Angle Class II malocclusion.The post-treatment changes in TMJ morphology were analyzed, based on TMJ cephalometric laminographs in 19 patients with Angle Class II malocclusion and labial inclination of the upper incisors after premolar extraction.The condylar pass angle, articular eminence to the Frankfort horizontal plane angle, and total, upper, and lower heights of the articular fossa increased significantly on both sides after treatment and retention. The anteroposterior width of the articular fossa decreased significantly on both sides after treatment and retention.These results suggest that adaptive bone remodeling of the TMJ occurs during the correction of occlusion with labial inclination of the upper incisors by orthodontic treatment after premolar extraction in patients with Angle Class II malocclusion.

To investigate the efficacy of using surgical glove (SG) compression therapy to prevent nanoparticle albumin-bound paclitaxel (nab-PTX)-induced peripheral neuropathy.Patients with primary and recurrent breast cancer who received 260 mg/mBetween August 2013 and January 2016, 43 patients were enrolled and 42 were evaluated. The occurrence rates of CTCAE grade 2 or higher sensory and motor peripheral neuropathies were significantly lower for SG-protected hands than for control hands (sensory neuropathy 21.4 vs. 76.1 %; motor neuropathy 26.2 vs. 57.1 %). No patients withdrew from this study because they could not tolerate the compression from the SGs. SG compression therapy significantly decreased the temperature of each fingertip by 1.6-2.2 °C as compared with the temperature before chemotherapy (p 0.0001).SG compression therapy is 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.

The CLEOPATRA trial reported the survival benefit of pertuzumab with trastuzumab plus docetaxel in HER2-positive metastatic breast cancer patients. However, there are a few case reports concerning the effects of a pertuzumab-containing regimen on brain metastases. A 55-year-old woman, who underwent curative surgery for breast cancer after neoadjuvant chemotherapy 5 years previously, developed repeated solitary brain metastasis in her right occipital lobe. Whole brain radiation therapy, stereotactic radiosurgery and 3 times of surgical resection were performed. Lapatinib and capecitabine plus tamoxifen were administered. The metastasis recurred in the stump of the previous surgery. Pertuzumab with trastuzumab plus docetaxel was initiated as second-line chemotherapy. A complete response of the brain metastasis was achieved, which persisted for 5 months. Pertuzumab with trastuzumab plus docetaxel was effective in reducing the brain metastases from breast cancer. Further studies are warranted to confirm the effect of this regimen on brain metastases.

Neoadjuvant chemotherapy (NAC) has been the standard therapy for breast cancer; however, whether sentinel lymph node biopsy (SLNB) should be performed after NAC in clinically node-positive patients is controversial. The indocyanine green (ICG)-fluorescence SLNB method (ICG-SLNB) provides a high identification rate as well as the ability to visualize lymphatic pathways, and previous studies have investigated the lymphatic pathways to sentinel lymph nodes (sentinel lymphatic pathways) using ICG- fluorescence method. In this study, we used ICG-SLNB to compare the sentinel lymphatic pathways before and after NAC to investigate whether they were affected by chemotherapy. Although the locations of the sentinel lymph nodes were unchanged, NAC did alter 42.8% of the sentinel lymphatic pathway routes. Our data indicate that SLNB after NAC can still detect the sentinel lymph nodes regardless of the presence or absence of metastasis. Since the false negative rates of SLNB after NAC for node-positive patients is higher than that for node-negative patients according to meta-analyses, further investigation is required to determine whether to perform SLNB after NAC for node-positive patients.

Among several resistant mechanisms of trastuzumab for HER2+ breast cancer patients, little is clear on resistant mechanism of trastuzumab mediated ADCC. 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. To establish the trastuzumab mediated ADCC resistant SK-BR-3, PBMCs are co-cultured with 1µg/ml of trastuzumab and SK-BR-3 for 4 hours. We removed CD45 positive PBMC by using MACS separation system and obtained ADCC resistant SK-BR-3. After we expanded the resistant cells and repeated these processes several times, we established the ADCC resistant SK-BR-3. The cytotoxicity of ADCC resistant SK-BR-3, which was evaluated by LDH assay, was significantly reduced as compared with that of normal SK-BR-3 (38.7% and 67.4%, respectively). To sort candidate genes for the resistance, we analyzed the gene expression profile of ADCC resistant SK-BR-3 by using DNA microarray and we focused on ATP6V1B1 gene that was significantly reduced on the resistant cells. 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. To evaluate the role of ATP6V1B1 on the trastuzumab mediated ADCC resistant mechanism, we established ATP6V1B1 knockout SKBR3 by using CRISPR/Cas9 system. We cultured from small numbers of the knock out cells and expand to obtain completely knock out cells. We confirmed that the ATP6V1B1 knockout cell line was knocked out by western blotting and immunohistochemistry. We found that ATP6V1B1-knockout SK-BR-3 were significantly less ADCC activity as compared to control SK-BR-3(67.4% and 21.2%, respectively). Furthermore, as the same as the result of ADCC activity, we found that ATP6V1B1-knockout SK-BR-3 was less AICC (antibody-independent cellular cytotoxicity) activity as compared to control SK-BR-3(41.2% and 12.5%, respectively). Based on the in vitro findings, we evaluated the role of ATP6V1B1 expression in HER2 positive breast cancer patients who are treated with neoadjuvant chemotherapy (NAC) containing trastuzumab. Biopsy samples before NAC were stained with ATP6V1B1 immunohistochemically. Four pathological complete response (pCR) cases after NAC and 4 non pCR cases were included. We found that non pCR cases showed significantly week expression of the ATP6V1B1 protein as compared to that of pCR cases. ATP6V1B1 gene is one of the isoforms of the Vacuolar (V)-ATPase which is located in intracellular membrane that regulate the cytosolic pH and membrane trafficking. It is hypothesized that ATP6V1B1 is involved in somewhere through the process of cytotoxicity mediated by perforin and granzymes and now we are trying to explore the mechanisms further. Citation Format: Mariko Nishie, Eiji Suzuki, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Takeshi Kotake, Masahiro Hirata, Fengling Pu, Keiko Sakamoto, Masakazu Hattori, Tomoharu Sugie, Masakazu Toi. Down regulation of ATP6V1B1 in HER2+ breast cancer leads resistance against trastuzumab mediated antibody dependent cellular cytotoxicity and it might be associated with less efficacy of trastuzumab clinically [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 4151. doi:10.1158/1538-7445.AM2017-4151

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

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

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.

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.

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.

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.

Chemotherapy-induced oral mucositis is a common adverse event in breast cancer patients. Breakdown of the mucosal barrier predisposes the patient to bacterial, fungal and viral superinfection, especially candidiasis. We demonstrated the frequency of chemotherapy-induced oral mucositis and oral candidiasis, and the efficacy of antimycotic agents in breast cancer patients. We investigated 32 patients with advanced and metastatic breast cancer who underwent chemotherapy in our department from March, 2009 to August, 2010. The chemotherapy regimens were as follows: FEC (epirubicin/5-FU/cyclophosphamide) followed by taxanes: 21, FEC: 1, TC (docetaxel/cyclophosphamide): 7, DOC (docetaxel): 3, and CPT-11/S-1: 1. Patients had blood and bacteria tests of the oral cavity at the time mucositis symptoms appeared. We administered an antimycotic agent (itraconazole) and evaluated its effect on mucositis at the time mucositis symptoms appeared. 56. 3% of patients had chemotherapy-induced oral mucositis, and 38. 9% of the mucositis patients had oral candidiasis. The incidence of mucositis increased when severe neutropenia occurred. 92. 9% of mucositis patients were cured or improved by itraconazole. In conclusion, chemotherapy caused oral candidiasis in 40% of cases with oral mucositis, and in about 56% of breast cancer patients. The antimycotic agent may be useful for chemotherapy-induced oral mucositis in breast cancer patients.

The benefit of neo-adjuvant chemotherapy for liver-limited metastatic colorectal cancer is still controversial. This study defined the resectability regardless of the size and number of liver metastases, and attempted curative hepatic resection in all cases.Sixty-four patients that tolerated chemotherapy were diagnosed with CLM (colorectal liver metastases) without extrahepatic metastase from January 2007 to November 2010, and received an oxaliplatin-based regimen. This study assessed the resectability after chemotherapy, and the patients were divided in two groups; the resected and unresected group. Sixteen patients underwent hepatic resection without chemotherapy.Thirty-five patients underwent surgical resection (resected group) and twenty-nine patients were considered unresectable (unresected group). All 35 patients in the resected group safely received oxaliplatin-based chemotherapy safely without serious adverse effects. No serious postoperative complications were observed. The median overall survival (MST) was significantly higher in the resected than in the unresected group (56.93 [95% CI 38.13-75.73] and 25.07 months [95% CI 17.87-32.26], respectively; P0.001). The median disease-free survival was 20.2 [95% CI 8.82-31.65] months in the resected group.Preoperative chemotherapy for CLM is well tolerated and does not increase postoperative complications. Curative surgery with preoperative chemotherapy has the potential to improve the overall survival in patients with CLM.

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 manufacturer's 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. Resultmean HV (ng/mL)mean EBC (ng/mL)mean MBC (ng/mL)EBC vs. HVEBC vs. HVMBC vs. HVMBC vs. HVMBC vs. EBCMBC vs. EBCp value95% CIp value95% CIp value95% CICD40.074460.90560.86520.2921-0.1404 to 0.43730.2728-0.08899 to 0.30520.6361-0.2114 to 0.1307CD81.2850.84181.1390.1896-1.129 to 0.24270.5976-0.7027 to 0.41070.0793-0.03641 to 0.6311CD400.70490.84941.0040.2892-0.1349 to 0.42390.0827-0.04087 to 0.63890.2334-0.1036 to 0.4127CD561.6372.4242.8750.4563-1.399 to 2.9740.2002-0.6881 to 3.1650.5711-1.144 to 2.046CD800.6761.4821.3250.0911-0.1445 yo 1.7560.0320.05925 to 1.2380.5747-0.7173 to 0.4036CTLA41.8273.0182.80.3655-1.519 to 3.9000.0918-0.1668 to 2.1120.746-1.567 to 1.131FOXP30.78011.0382.2960.4605-0.4647 to 0.98020.02780.1760 to 2.8570.01320.2776 to 2.240IDO11.0251.9841.4250.2317-0.6764 to 0.26120.3982-0.5508 to 1.3500.2437-1.516 to 0.3971IDO20.56780.35280.37990.3447-0.6912 to 0.26120.2673-0.5304 to 0.15450.8255-0.2222 to 0.2764NKG2D1.5421.1852.040.3976-1.228 to 0.51380.3432-0.5553 to 1.5520.03730.05292 to 1.658NRP11.8614.4371.1560.1359-0.9076 to 6.0580.3538-1.455 to 3.9470.2782-3.780 to 1.121PD-10.53520.35270.40090.1798-0.4582 to 0.093290.3895-0.4472 to 0.17870.6692-0.1783 to 0.2747PD-L10.87321.0372.2180.7193-0.7861 to 1.1140.03670.08793 to 2.6020.01730.2197 to 2.142 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

We and others previously reported that HER2 was transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis. In the current study, we report that 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 antibody administration. Interestingly, the EGFR-trogocytosed MDA-MB-231 cells reduced the expression of EGFR and changed their morphology to the spindle shape (well known as mesenchymal transition) and acquired greater cell motility. These cancer cell properties are representative as higher malignant potentials in breast cancer patients. Thus, we hypothesized that EGFR expression on tumor infiltrating lymphocytes (TILs), which probably is caused via EGFR trogocytosis by TILs, is correlated with higher tumor grade. We evaluated the EGFR expression of TILs in 36 triple negative breast cancer specimens and found the correlation between EGFR expression status on TILs and higher tumor grade (P = 0.02). These findings may explain the reason why triple negative breast cancer with greater proportion of TILs exhibit higher tumor grade and poor prognosis. Citation Format: Eiji Suzuki, Ayane Yamaguchi, Tatsuki R. Kataoka, Masahiro Hirata, Kosuke Kawaguchi, Mariko Nishie, Masakazu Toi. EGFR is transferred from triple negative breast cancer cells to immune cells via trogocytosis and expression of EGFR on immune cells is associated with high tumor grade of triple negative 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 2349. doi:10.1158/1538-7445.AM2015-2349

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.

164 Background: Neoadjuvant chemotherapy (NAC) is a standard therapy for breast cancer. However, the changes in the lymphatic route to the sentinel lymph node (SLN) before and after NAC are unclear. Indocyanine green (ICG) fluorescence method of SLN biopsy (ICG-SLNB) is useful because of its high identification rate of the SLN and ability to visualize lymphatic routes. Using ICG-SLNB, we compared the lymphatic routes to the SLN (sentinel lymphatic routes) before and after NAC and investigated whether chemotherapy affects the sentinel lymphatic routes. Methods: From October 2010 to April 2012 20 patients received NAC. In 13 cases of clinical node-negative cancer, we performed SLNB before NAC. We recorded the sentinel lymphatic routes before NAC and confirmed the routes at surgery after NAC by using the same method. In 7 cases of clinical node-positive cancer, we recorded the localization of SLN and lymphatic routes by using the ICG fluorescence method before NAC. After NAC, we performed SLNB with axillary dissection and recorded the sentinel lymphatic routes by photography and drawing. Results: Regardless of the presence or absence of lymph node metastasis, the SLN and sentinel lymphatic routes could be detected before and after NAC in all cases. All sentinel lymphatic routes reached the same location of the SLN before and after NAC. In 3 cases, the number of sentinel lymphatic routes had increased after NAC, but the location of the SLN was the same before and after NAC. Other cases showed no changes in the sentinel lymphatic routes. In clinical node-positive cases of pathological complete response, there was no metastasis to the SLN, but there were 2 metastases in the lymph nodes after back-up dissection. Conclusions: ICG fluorescence method provided visual confirmation that the sentinel lymphatic routes were not affected by NAC. This result suggests that SLNB after NAC can be used to safely and positively identify the SLN in clinical node-negative patients. Further investigation would be required to safely perform SLNB after NAC in clinical node-positive patients.

465 Background: Recently, several papers have reported the advantage of neoadjuvant chemotherapy for liver limited metastatic colorectal cancer. In these papers, most study groups used criteria for non-resectability due to size and/or number of metastases. Our criteria for resectability of colorectal liver metastases (CLM) depends on the size of remnant liver volume (>30%) and expected function after the removal of all metastases. Then, we assessed the feasibility and potential benefits of chemotherapy administered before surgery to patients with CLM retrospectively. Methods: From January 2007 to April 2010, 67 chemotherapy-naive patients were diagnosed as CLM without extra-hepatic metastases. After chemotherapy, we assessed the resectability with radiological examination.Then, each case was divided in two groups, resected group and unresected group. Overall survival, median disease-free survival and postoperative complications were analyzed. Results: 63 patients received oxaliplatin-based combination regimen and 4 patients other regimen. 30 patients (resected group) received R0 resection and 37 patients (unresected group) were considered as unresectable. No serious postoperative complications were observed. Overall survival was significantly higher in resected group than in unresected group (42.3 month and 29.1 month; P Conclusions: Intensive treatment with neoadjuvant chemotherapy for CLM is well tolerated. Curative surgery improved significantly overall survival in patients with CLM. Neoadjuvant chemotherapy such as mFOLFOX6 may lead to increased resectability but do not increase postoperative complications. For most patients with resectable CLM, neoadjuvant chemotherapy should be considered the standard treatment. No significant financial relationships to disclose.