Kenta Kawahara

Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS

Purpose: The peptides derived from ideal cancer–testis antigens, including LY6K, CDCA1, and IMP3 (identified using genome-wide cDNA microarray analyses), were used in immunotherapy for head and neck squamous cell cancer (HNSCC). In this trial, we analyzed the immune response to and safety and efficacy of vaccine therapy. Experimental Design: A total of 37 patients with advanced HNSCC were enrolled in this trial of peptide vaccine therapy, and the OS, PFS, and immunologic response were evaluated using enzyme-linked ImmunoSpot (ELISPOT) and pentamer assays. The peptides were subcutaneously administered weekly with IFA. The primary endpoints were evaluated on the basis of differences between HLA-A*2402-positive [A24(+)] patients treated with peptide vaccine therapy and –negative [A24(−)] patients treated without peptide vaccine therapy among those with advanced HNSCC. Results: Our cancer vaccine therapy was well tolerated. The OS of the A24(+) vaccinated group (n = 37) was statistically significantly longer than that of the A24(−) group (n = 18) and median survival time (MST) was 4.9 versus 3.5 months, respectively; P < 0.05. One of the patients exhibited a complete response. In the A24(+) vaccinated group, the ELISPOT assay identified LY6K-, CDCA1-, and IMP3-specific CTL responses in 85.7%, 64.3%, and 42.9% of the patients, respectively. The patients showing LY6K- and CDCA1-specific CTL responses demonstrated a longer OS than those without CTL induction. Moreover, the patients exhibiting CTL induction for multiple peptides demonstrated better clinical responses. Conclusions: The immune response induced by this vaccine may improve the prognosis of patients with advanced HNSCC. Clin Cancer Res; 21(2); 312–21. ©2014 AACR.

Identification of promiscuous KIF20A long peptides bearing both CD4+ and CD8+ T-cell epitopes: KIF20A-specific CD4+ T-cell immunity in patients with malignant tumor

Purpose: To identify long peptides (LP) derived from a novel tumor-associated antigen (TAA), kinesin family member 20A (KIF20A), which induce tumor-specific T-helper type 1 (TH1) cells and CTLs. Experimental Design: We combined information from a recently developed computer algorithm predicting HLA class II–binding peptides with KIF20A-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate promiscuous TH1-cell epitopes containing CTL epitopes. Peripheral blood mononuclear cells (PBMC) derived from healthy donors or patients with head-and-neck malignant tumor (HNMT) were used to study the immunogenicity of KIF20A-LPs, and the in vitro cross-priming potential of KIF20A-LPs bearing CTL epitopes. We used HLA-A24 transgenic mice to address whether vaccination with KIF20A-LP induces efficient cross-priming of CTLs in vivo. The TH1-cell response to KIF20A-LPs in HNMT patients receiving immunotherapy with TAA-derived CTL-epitope peptides was analyzed using IFN-γ enzyme-linked immunospot assays. Results: We identified promiscuous KIF20A-LPs bearing naturally processed epitopes recognized by CD4+ T cells and CTLs. KIF20A-specific CTLs were induced by vaccination with a KIF20A-LP in vivo. KIF20A expression was detected in 55% of HNMT by immunohistochemistry, and significant frequencies of KIF20A-specific TH1 cell responses were detected after short-term in vitro stimulation of PBMCs with KIF20A-LPs in 50% of HNMT patients, but not in healthy donors. Furthermore, these responses were associated with KIF20A expression in HNMT tissues. Conclusions: These are the first results showing the presence of KIF20A-specific TH1 cell responses in HNMT patients and underline the possible utility of KIF20A-LPs for propagation of TH1 cells and CTLs. Clin Cancer Res; 19(16); 4508–20. ©2013 AACR.

Overexpression of cIAP2 contributes to 5-FU resistance and a poor prognosis in oral squamous cell carcinoma

Background:Resistance to 5-fluorouracil (5-FU) is a major obstacle in treating oral squamous cell carcinoma (OSCC). However, little is known about apoptosis resistance, which contributes to 5-FU resistance in OSCC.Methods:We focussed on the cellular inhibitor of apoptosis protein 2 (cIAP2) on the basis of a DNA microarray data using parental and 5-FU-resistant OSCC cell lines. The effects of cIAP2 downregulation on 5-FU sensitivity and apoptosis were evaluated. An immunohistochemical analysis of cIAP2 and related proteins, cIAP1 and X-linked IAP, was performed in 54 OSCC patients who were treated with 5-FU-based chemoradiotherapy and surgery.Results:The downregulation of cIAP2 significantly enhanced the sensitivity of the 5-FU-resistant cells to 5-FU, with a significant increase in apoptosis. The immunohistochemical analysis demonstrated a high cIAP2 tumour expression to significantly correlate with the pathological response to chemoradiotherapy. Furthermore, a Cox regression analysis revealed the cIAP2 expression status (hazard ratio, 4.91; P=0.037) and the pathological response to chemoradiotherapy (hazard ratio, 0.418; P=0.016) to be significant prognostic factors for OSCC patients.Conclusion:These novel findings demonstrate that cIAP2 may represent a potentially useful therapeutic target for improving the treatment and survival of OSCC patients, particularly in the setting of 5-FU resistance.

Overexpression of fibronectin confers cell adhesion‑mediated drug resistance (CAM-DR) against 5-FU in oral squamous cell carcinoma cells

The tumor-associated microenvironment has been shown to protect tumor cells from treatment, and the extracellular matrix (ECM) is known to affect drug resistance as a key regulator of the tumor microenvironment. However, little is known about cell adhesion-mediated drug resistance (CAM-DR) due to cell-ECM contact in patients with oral squamous cell carcinoma (OSCC). In the present study, we evaluated the ECM molecule fibronectin (FN) using DNA microarray data obtained from parental and 5-FU-resistant OSCC cell lines. We investigated the effects of cell adhesion to FN on 5-FU resistance in OSCC cells and examined the activation of FN receptor β1 integrin-mediated survival regulators such as ILK, Akt and NF-κB. In addition, we investigated whether FNIII14, a 22-mer peptide derived from FN that potently prevents β1 integrin-mediated adhesion to FN, could overcome CAM-DR against 5-FU in OSCC cells and examined the activation of survival regulators and apoptosis-related molecules. Consequently, we obtained the following results. FN was extracellularly overexpressed in the 5-FU-resistant cells compared with that observed in the 5-FU-sensitive cells. Cell adhesion to FN enhanced 5-FU resistance and activated integrin-mediated ILK/Akt/NF-κB survival signaling in the 5-FU-resistant OSCC cells. Furthermore, the inhibition of cell adhesion to FN by FNIII14 enhanced chemosensitivity to 5-FU and apoptosis by suppressing ILK/Akt/NF-κB signaling in the 5-FU-resistant cells. These novel findings demonstrate that FN is a potentially useful biomarker and therapeutic target for improving the treatment of OSCC, particularly in the setting of 5-FU resistance.

Pre-treatment neutrophil to lymphocyte ratio predicts the chemoradiotherapy outcome and survival in patients with oral squamous cell carcinoma: a retrospective study

BackgroundThe Neutrophil to lymphocyte ratio (NLR) has prognostic value in patients with a variety of cancers. However, its prognostic significance in oral squamous cell carcinoma (OSCC) patients has not been fully explored. The purpose of this study was to determine the clinical significance of NLR in patients with OSCC.MethodsOSCC patients who underwent surgery following 5-fluorouracil (5-FU)-based chemoradiotherapy were enrolled in this study. The associations between the NLR status and various clinicopathological features were examined, and the effects of the NLR on the prognosis were evaluated. Analysis of circulating interleukin-6 (IL-6) was carried out and correlation with NLR and C-reactive protein concentration (CRP) was examined.ResultsAn elevated NLR was significantly correlated with advanced T-stage and poor response to chemoradiotherapy. Moreover, a Cox regression analysis based on the disease-free survival (DFS) revealed the NLR status (hazard ratio, 2.013; P = 0.041) and pathological response to chemoradiotherapy (hazard ratio, 0.226; P = 0.001) to be significant prognostic factors in OSCC patients. Furthermore, circulating IL-6 was found to correlate with NLR and CRP.ConclusionThe NLR is a potential biomarker for predicting the clinical response to 5-FU-based chemoradiotherapy and the survival in OSCC patients, and the systemic inflammatory response may be potential target for improving patient’s prognosis.

Selective inhibition of nuclear factor-κB by nuclear factor-κB essential modulator-binding domain peptide suppresses the metastasis of highly metastatic oral squamous cell carcinoma

Nuclear factor‐κB (NF‐κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF‐κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP‐expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF‐κB activities and expression of NF‐κB‐regulated metastasis‐related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor‐engrafted mice were monitored. Furthermore, the effects of a selective NF‐κB inhibitor, NEMO‐binding domain (NBD) peptide, on metastasis in GSAS/N5‐engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF‐κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF‐κB‐regulated metastasis‐related molecules, such as fibronectin, β1 integrin, MMP‐1, ‐2, ‐9, and ‐14, and VEGF‐C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5‐inoculated mice, and was accompanied by the downregulation of the NF‐κB‐regulated metastasis‐related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF‐κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC. (Cancer Sci 2012; 103: 455–463)

The tumour stromal features are associated with resistance to 5‐FU‐based chemoradiotherapy and a poor prognosis in patients with oral squamous cell carcinoma

It has been increasingly recognized that the tumour microenvironment is a critical factor involved in cancer progression. However, little is known about the clinical value of the stromal features in oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the clinical significance of cancer‐associated fibroblasts (CAFs) and tumour‐associated macrophages (TAMs) in OSCC. OSCC specimens were obtained from 60 patients who underwent surgery following 5‐fluorouracil‐based chemoradiotherapy. Paraffin‐embedded sections obtained from biopsy specimens were immunohistochemically analysed. The associations among CAFs, TAMs and various clinicopathological features were examined, and the effects of CAFs and TAMs on the prognosis were evaluated. In the group with a high level of CAFs, the incidence of advanced pT‐ and pN‐stage cases was significantly higher than that in the group with the low level. A high TAMs tumour expression was significantly correlated with a poor response to preoperative chemoradiotherapy. A Kaplan–Meier analysis revealed that higher numbers of CAFs and TAMs were significantly correlated with a poor prognosis. These findings suggest that TAMs are a potential biomarker for predicting the clinical response to 5‐FU‐based chemoradiotherapy, and the expression status of the CAFs and TAMs may be useful for making treatment decisions to improve the survival of OSCC patients.

Overexpression of nucleostemin contributes to an advanced malignant phenotype and a poor prognosis in oral squamous cell carcinoma.

Background:Nucleostemin (NS) is essential for the maintenance of stem cell properties, the functions of which remain poorly understood in cancer cells. The purpose of this study was to explore the impact of NS on malignancy and its clinical significance in oral squamous cell carcinoma (OSCC) patients.Methods:We investigated the effects of NS on the proliferation and invasion of OSCC using NS-overexpressing or -knockdown OSCC cells. We assessed the activation of the STAT3 (signal transducer and activator of transcription 3) signalling pathway and the downstream targets in the cells with different expression levels of NS. An immunohistochemical analysis of NS was also performed in 54 OSCC patients who were treated with preoperative chemoradiotherapy and surgery.Results:The overexpression of NS significantly enhanced the proliferation and invasive potential of OSCC cells. On the other hand, downregulation of NS suppressed the invasiveness of the cells. The alterations of these malignant phenotypes were associated with the activation of STAT3 signalling and its downstream targets. An immunohistochemical analysis demonstrated that a high NS tumour expression level significantly correlated with an advanced T-stage and N-stage. Furthermore, a Cox regression analysis revealed that the NS status (hazard ratio, 9.09; P=0.002) was a significant progression factor for OSCC patients.Conclusions:Our results suggest that targeting NS may provide a promising treatment for highly malignant OSCC.

IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma

Background:In promoting tumour malignancy IL-6 signalling is considered to have an important role. However, the biological roles of IL-6 on radiosensitivity in oral squamous cell carcinoma (OSCC) remain largely unclear. The objective of this study is to determine the effects and molecular mechanisms of IL-6 on radiosensitivity in OSCC.Methods:Two OSCC cell lines, and OSCC tissue samples with radioresistant cells were used. We examined the effects of IL-6, or tocilizumab, a humanised anti-human IL-6 receptor antibody, or both on radiosensitivity and DNA damage after X-ray irradiation in vitro. In addition, we investigated the involvement of the Nrf2-antioxidant pathway in IL-6-mediated radioresistant mechanisms using OSCC cell lines and tissues.Results:Increased levels of IL-6 suppressed radiation-induced cell death, and the blockade of IL-6 signalling by tocilizumab sensitised tumour cells to radiation. The radioresistant effect of IL-6 was associated with decreased DNA damage after radiation. We also found that IL-6 promotes the activation of not only the downstream molecule STAT3 but also the Nrf2-antioxidant pathway, leading to a significant decrease in oxidative stress by upregulating Mn-SOD.Conclusions:These results indicate that the blockade of IL-6 signalling combined with conventional radiotherapy could augment the treatment response and survival rate in patients with radioresistant OSCC.

Angiosarcoma of the oral cavity: a clinicopathological study and a review of the literature

Soft tissue sarcomas are uncommon in the head and neck. Primary angiosarcomas of the oral cavity area are extremely rare, and have mostly been presented as case reports. This paper presents the clinical and histological features of three such cases. All patients were diagnosed based on the presence of rapidly extending masses involving the tongue, maxillary gingiva, or mandibular gingiva; bone destruction was present in two cases. The resected specimens revealed clustered large, pleomorphic, and spindle-shaped cells with a markedly haemorrhagic background. Tumour cells showed expression of vascular endothelial markers, such a CD31, CD34, and factor VIII-related antigen. Despite undergoing radical surgery, distant metastases developed in all three cases. We also studied the clinicopathological features of a series of oral angiosarcomas. This article therefore reports the clinicopathological features of the three new cases and provides a review of the cases of primary oral angiosarcoma reported during the past 20 years.