Moe Tsuda

Effects of Cryotherapy on Objective and Subjective Symptoms of Paclitaxel-Induced Neuropathy: Prospective Self-Controlled Trial

Abstract Background Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting and disabling side effect of taxane anticancer agents. We prospectively evaluated the efficacy of cryotherapy for CIPN prevention. Methods Breast cancer patients treated weekly with paclitaxel (80 mg/m2 for one hour) wore frozen gloves and socks on the dominant side for 90 minutes, including the entire duration of drug infusion. Symptoms on the treated sides were compared with those on the untreated (nondominant) sides. The primary end point was CIPN incidence assessed by changes in tactile sensitivity from pretreatment baseline in a monofilament test at a cumulative dose of 960 mg/m2. We also assessed thermosensory deficits, subjective symptoms (Patient Neuropathy Questionnaire [PNQ]), manipulative dexterity, and the time to events and hazard ratio by PNQ. All statistical tests were two-sided. Results Among the 40 patients, four did not reach the cumulative dose (due to the occurrence of pneumonia, severe fatigue, severe liver dysfunction, and macular edema), leaving 36 patients for analysis. None dropped out due to cold intolerance. The incidence of objective and subjective CIPN signs was clinically and statistically significantly lower on the intervention side than on the control (hand: tactile sensitivity = 27.8% vs 80.6%, odds ratio [OR] = 20.00, 95% confidence interval [CI] = 3.20 to 828.96, P < .001; foot: tacile sensitivity = 25.0% vs 63.9%, OR = infinite, 95% CI = 3.32 to infinite, P < .001; hand: warm sense = 8.8% vs 32.4%, OR = 9.00, 95% CI = 1.25 to 394.48, P = .02; foot: warm sense: 33.4% vs 57.6%, OR = 5.00, 95% CI = 1.07 to 46.93, P = .04; hand: PNQ = 2.8% vs 41.7%, OR = infinite, 95% CI = 3.32 to infinite, P < .001; foot: PNQ = 2.8% vs 36.1%, OR = infinite, 95% CI = 2.78 to infinite, P < .001; hand: hazard ratio [HR] = 0.13, 95% CI = 0.05 to 0.34; foot: HR = 0.13, 95% CI = 0.04 to 0.38, dexterity mean delay = −2.5 seconds, SD = 12.0 seconds, vs + 8.6 seconds, SD = 25.8 seconds, P = .005). Conclusions Cryotherapy is useful for preventing both the objective and subjective symptoms of CIPN and resultant dysfunction.

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

Abstract P2-11-07: Duration of fasting before taking lapatinib is associated with skin toxicity in neoadjuvant treatment of HER2 positive breast cancer: A cohort study from JBCRG-16/Neo-LaTH

Background : In neoadjuvant dual HER2 blockade, over 30% of patients fail to complete treatment as planned because of lapatinib-induced diarrhea, rash, and hepatotoxicity. Lapatinib bioavailability, which affects both efficacy and toxicity, is influenced by prandial conditions. Methods : To investigate the association between lapatinib dosage timing and toxicity, we reviewed the medical records of patients who were enrolled in the JBCRG-16/Neo-LaTH randomized phase II multicenter trial evaluating the efficacy and safety of neoadjuvant 1000 mg/day lapatinib (La) and trastuzumab (T) therapy for 6 or 12 weeks followed by 750 mg/day La, T and weekly paclitaxel for 12 weeks in Japanese patients with primary HER2 positive breast cancer. Lapatinib dosage timing was divided into three groups: after overnight fasting, between meals, and at bedtime. We also measured serum lapatinib concentrations at steady state and dosage timing on the day prior to pharmacokinetic blood sampling. The primary endpoint was to investigate the association between lapatinib dosage timing and frequency of ≥grade 2 diarrhea. The secondary endpoint was to assess the association between dosage timing and other toxicities, pharmacokinetics, efficacy, and treatment discontinuation. Statistical analyses performed included one-way ANOVA, Welch9s test and logistic regression. Results : Out of 213 patients enrolled in JBCRG-16/Neo LaTH, we obtained dosage timing data from 143 (67%) patients: 16 (11%) after overnight fasting, 53 (37%) between meals, and 74 (52%) at bedtime. Serum lapatinib concentrations were obtained in 34/143 (24%) of patients. Dosage timing was not associated with ≥grade 2 diarrhea (8/16 (50%) after overnight fasting, 18/53 (34%) between meals, and 26/74 (35%) at bedtime; p = 0.48). However, multivariate analysis revealed that the after overnight fasting group is less likely to develop acne-like rash during La + T treatment regardless of age, BMI, or treatment. In addition, serum lapatinib trough concentration and it9s variability were significantly reduced in the after overnight fasting group (mean ± standard deviation (SD) = 0.35 ± 0.15 µg/ml, coefficient of variation (CV) = 42.7%) as compared to the others (mean ± SD = 0.77 ± 0.44 µg/ml, CV = 57.8%) (p p = 0.79). Conclusions : These data suggest that overnight fasting stabilizes the bioavailability of lapatinib, which may aid in managing lapatinib-induced rash without diminishing its therapeutic efficacy. Citation Format: Tsuda M, Ishiguro H, Toriguchi N, Masuda N, Bando H, Ohgami M, Homma M, Morita S, Yamamoto N, Kuroi K, Takano T, Shimizu S, Toi M. Duration of fasting before taking lapatinib is associated with skin toxicity in neoadjuvant treatment of HER2 positive breast cancer: A cohort study from JBCRG-16/Neo-LaTH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-11-07.

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.

O2-19-5The effects of icing on paclitaxel-induced peripheral neuropathy among breast cancer patients: a self-controlled trial

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a common, dose-limiting and disabling side effect with no proven effective treatment or prevention. Therefore, we aimed to evaluate the effectiveness of frozen glove and sock (FGS) in preventing CIPN with multiple assessments. Methods: Forty breast cancer patients treated weekly with paclitaxel (80 mg/m for 1 h) wore FGS on the dominant hand and foot for 90 min, including the entire duration of drug infusion. Signs and symptoms on the treated sides were compared to the untreated (nondominant) sides. Primary endpoint was CIPN incidence as assessed by tactile sensitivity change from pretreatment baseline in monofilament test at a cumulative dose of 960 mg/m (12 administrations). We also assessed thermosensory deficits, subjective symptoms (Patient Neuropathy Questionnaire (PNQ)), manipulative dexterity, and the cumulative dose-subjective events association. McNemar’s test (tactile, thermal, and PNQ), log-rank test (cumulative dose-event relation), and paired t-test (dexterity) were used for statistical comparisons.

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 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.