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Concerns about off-target effects (OTEs) of genomic DNA cleavages by gene-editing enzymes have been raised, especially for OTEs that go undetected due to technical limitations. Since no explicit guidelines have been in place for risk assessment of OTEs, the regulatory authorities' concept of an acceptable evaluation scheme for OTEs in the investigational drug application (IND) has not been clear. Here, we clarify the regulatory expectations by examining reports on OTE evaluations of leading gene-editing products that have achieved IND clearance.We collected and analyzed the gene-editing products that have entered clinical trials by searching on ClinicalTrials.gov and EU-Clinical-Trial-Registries, and related reports for OTE evaluations from Google Scholar, PubMed, and the developers' websites.We found a common two-step verification method used for different products at the preclinical stage. First, numerous potential off-target loci (POLs) are listed with state-of-the-art high-sensitivity detection methods and theoretical screens; Second, these OTEs are checked by amplicon sequencing of the POLs after treatment by enzymes in in vitro models close to clinical use conditions. Only the OTEs that can be detected and verified are addressed in the risk assessment in the translational phase from preclinical to clinical study.Here, we describe a clear scheme for risk assessment of OTEs at the key translational phase, based on the common features in protocols for gene-editing products that were cleared for use in clinical trials. This report will provide a guide for those newly attempting to conduct clinical development in this field.

Purpose: The prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and there are only a few standard treatments. We conducted a phase I trial to investigate the safety, immune response, and antitumor effect of vaccination with four peptides derived from cancer-testis antigens, with a focus on their fluctuations during long-term vaccination until the disease had progressed.Experimental Design: Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. HLA-A*2402–restricted epitope peptides, lymphocyte antigen 6 complex locus K, TTK protein kinase, insulin-like growth factor-II mRNA-binding protein 3, and DEP domain containing 1 were vaccinated subcutaneously once a week at doses of 0.5, 1, or 2 mg and continued until disease progression. The adverse events were assessed by Common Terminology Criteria for Adverse Events and the immune response was monitored by an enzyme-linked immunospot assay or by flow cytometry. The clinical effects observed were tumor response, progression-free survival (PFS), and overall survival (OS).Results: Four-peptide vaccination was well tolerated. No grade 3 or 4 adverse events were observed. Peptide-specific T-cell immune responses were observed in seven of nine patients and clinical responses were observed in six of nine patients. The median PFS and OS were 156 and 380 days. The injection site reaction and CTL induction seemed to be prognostic factors of both PFS and OS.Conclusions: Four-peptide vaccination was well tolerated and seemed to provide some clinical benefit to some patients. These immunologic and clinical responses were maintained over the long term through continuous vaccinations. Clin Cancer Res; 19(8); 2224–31. ©2013 AACR.

PDF File - 111K, Immunologic monitoring of each peptide-specific T cell response in each patient.

PDF File - 23K, Legends for Figures 1 and 2.

PDF File - 125K, An algorithm to define the responses of antigen-specific T cells by using the ELISPOT assay.

Background: Demographic, healthcare resource utilization (HCRU), and healthcare costs of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) patients, and those progressed to liver cirrhosis (LC) and hepatocellular carcinoma (HCC) was assessed from Japan Medical Data Center (JMDC) claims database. Methods: 185,012 NAFLD or NASH patients between 1st January 2006 and 31st July 2018 were identified from JMDC database. Mean annual all-cause HCRU and costs were reported pre- and post-index date of the initial diagnosis of NAFLD/NASH, LC and HCC. Age, gender, and comorbidity influence on post-index cost was assessed with a multivariable analysis. Results: 123,379 NAFLD/NASH, 205 LC, and 96 HCC patients were selected into 3 liver severity cohorts based on the study inclusion/exclusion criteria. Majority of patients were male (70.0% to 71.7%) with comorbid hyperlipidaemia (50.2% to 61.5%), diabetes mellitus (40.0% to 60.4%), and hypertension (27.9 % to 42.7%) most prevalent across the 3 liver disease states. The mean all-cause healthcare annual cost significantly increased with liver disease severity, at ¥146,096, ¥399,425 and ¥842,875 for NAFLD/NASH, LC and HCC patients, respectively. Comorbid cardiovascular disease (CVD), following by type 2 diabetes mellitus (T2DM) had the highest influence on all-cause cost with a cost ratio of 1.77 and 1.45, respectively. Conclusions: NAFLD/NASH patients with LC or HCC have increased comorbidity burden, high HCRU, and healthcare costs, which further highlights the importance and need of timely identification and management for NAFLD and NASH patients in Japan.

Oncolytic virus therapy (OVT) represents a new class of therapeutic agents in cancer treatment. The molecular and cellular mechanisms of action of OVTs have been evaluated in nonclinical/clinical phase trials. Various genetically modified viruses have been developed as oncolytic agents, and the first approval of an OVT for clinical use was issued by the US Food and Drug Administration in 2015. In this context, more and more clinical development of OVTs is anticipated in the future. This article provides a risk assessment of OVT based on the safety data obtained from all clinical trials to date using a publicly available database. The most common adverse events (AEs) observed in clinical trials have been infection-related symptoms such as fatigue, chills, fever, and nausea; few serious AEs have been observed, regardless of the kind of virus or transfected genes. In vivo systemic infusion of OVTs demonstrated a high percentage of AEs, but most AEs were manageable using common drugs. This paper describes OVTs' specific safety/toxicity profiles and encourages the performance of further clinical trials of OVTs to address the most serious challenges anticipated in the development of OVTs as a new class of drugs for the treatment of cancer.

Green-emitting Tb3+-doped pollucite (Cs1-3xTbx)AlSi2O6 (x = 0.01–0.13) phosphors were synthesized by the high-temperature solid-state reaction method. Structural characterization of the prepared (Cs1-3xTbx)AlSi2O6 phosphors was performed using powder X-ray diffraction and X-ray photoelectron spectroscopy. The excitation and emission spectra showed green emission at ~543 nm because of the 5D4→7F5 transition under excitation at 238 nm. The optimum doping concentration of the Tb3+ ions was x = 0.07, and the quantum efficiency was 83.6%. The temperature dependence of emission spectra for x = 0.07 revealed that 92.7% and 78.4% of the initial value of the emission intensity at 300 K are retained at 400 K and 500 K, respectively. These results show that the green-emitting phosphors synthesized herein have good performance and potential applications in ultraviolet-C excited white light-emitting diodes.

We previously conducted a phase I clinical trial combining the HLA-A*2402-restricted KIF20A-derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single-armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1-year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide-specific immune responses. All enrolled patients received therapy without the HLA-A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1-year survival rates between the HLA-A*2402-matched and -unmatched groups were not significantly different. In the HLA-A*2402 matched group, patients showing peptide-specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA-A*2402-matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application.

Eu2+-doped CsAlSi2O6 (pollucite) phosphors were synthesized via high-temperature solid-state reaction to produce a white-light-emitting phosphor under UV irradiation. The obtained phosphors were characterized using X-ray diffraction, photoluminescence (PL), PL excitation, thermal stability, and PL decay. In contrast to previous reports, the fabricated phosphors emitted either white light or bluish white light with less thermal quenching and a higher internal-quantum efficiency (IQE) upon deep-UV excitation than upon near-UV excitation. The phosphors were subjected to shock compression at a pressure of 23 GPa to investigate the structural response of the pollucite. Although the pressure was higher than that required for a structural transition, no trace of a phase transition was observed. Although the chromaticity coordinates and thermal quenching of the shocked pollucite phosphors showed little change, a considerable degree of strain was induced and the PL intensity and IQE decreased due to a degradation in crystallinity.

A series of Eu2+ substituted pollucite [(Cs1-xEu1-2x)AlSi2O6 (CASO:xEu) (x = 0.001–0.08)] phosphors were synthesized by a high-temperature solid-state reaction method in a reducing atmosphere and characterized by X-ray diffraction, photoluminescence (PL), and PL excitation (PLE) measurements. Under excitation at 275 nm, CASO:xEu phosphors exhibit an emission band that shifts from the bright blue region with color coordinates of (0.159, 0.247) to the bright blue-green region with color coordinates of (0.225, 0.421) with increasing x. Such tunable luminescence color was attributed to the crystal field splitting effect of Eu2+ ions because the substitution of Eu2+ created vacancies at Cs sites, and fluctuations occurred at the positions occupied by the Eu2+ ions. Studies on the temperature dependence of luminescence for x = 0.01, 0.03 0.05, and 0.07 in CASO:xEu reveal that 92.5%, 85.3%, 74.9%, and 79.8% of the initial value of the luminescence intensity at 300 K are retained at 400 K, respectively. Tuning the emission spectra was possible by varying the value of x while maintaining a high thermal stability.

In 2012, the US Food and Drug Administration (FDA) issued the draft guidance “Determining the Extent of Safety Data Collection Needed in Late Stage Premarket and Postapproval Clinical Investigations.” The selective data collection approach proposed in this guidance leads to reductions in costs and work time and may improve the quality of the database for clinical trials. The current study evaluated the applicability of selective data collection for oncology drugs. The labeling information of oncology drugs obtained supplemental approvals from the FDA between 2005 and 2014 were used. The frequency of adverse reactions observed in clinical trials between the first approval and supplemental approvals of a specific drug were compared. Paired studies were categorized into the following 4 groups: A, same tumor type and same usage; B, same tumor type and different usage; C, different tumor type and same usage; D, different tumor type and different usage. A total of 46 study pairs for additional drug indications were investigated. In group A, 6 of the 7 pairs showed a high correlation coefficient (r = 0.988, 0.953, 0.947, 0.935, 0.853, and 0.846). Selective data collection should be adopted in cases in which the additional indication of a drug is for the same tumor type and usage as the first or previous indication.

Compassionate use, also called expanded access, provides an important pathway for patients with life-threatening conditions to gain access to unapproved investigational drugs, biologics and medical devices. Although the United States (US) and the countries of the Europe Union (EU) have mechanisms that are associated with the use of unapproved products, as of May 2015 there was no such mechanism in Japan. Instead, unapproved products are used under a physician's discretion in conjunction with the Japan Medical Practitioners' Act or Advanced Medical Care B. However, there are some issues and questions to consider under the current circumstances in Japan as follows: (A) it is difficult for the local regulator to monitor the use of unapproved products; (B) there is no information collected on the safety of these products to protect patients; (C) it is difficult to assure the quality of the products; (D) it is difficult for patients to obtain detailed information about unapproved products and their availability; and (E) it is not clear who should cover the cost of the unapproved products.In this paper, we assess the current compassionate use, or expanded access-related mechanisms, of the US, the EU and Japan in regard to drugs, medical devices and biologics, including human cells and tissue products, and discuss the benefits and issues of these mechanisms. The purpose of these mechanisms is principally to save patients with life-threatening condition. However, the information obtained after the compassionate use is potentially useful to facilitate marketing authorization. In fact, the data from compassionate use cases are employed in some approval review reports to indicate the product safety.

The aim of this study was to identify the differences between Japan and the United States in the approval status and regulatory actions for radiopharmaceuticals. The package inserts of radiopharmaceuticals that have been approved in the United States and Japan were used to investigate the status of approval in each country, examining the similarities and differences thereof and taking the regulatory systems of the 2 countries into account. Results revealed that since 1995, fewer products have been approved in Japan than in the United States: 16 radiopharmaceuticals have been approved in the United States, compared to only 7 in Japan. Although the clinical evaluation methodology for radiopharmaceuticals is still being developed, study results suggest that international harmonization of the regulatory systems and manufacturing controls for positron emission tomography drugs will be important for the development of such drugs in multinational countries, including Japan.

Background/Aim: MK615 extracted from Prunus mume was reported to have anti-inflammatory effects. In this article, we examined the in vivo antitumor effect of MK615 (an extract from Japanese apricot) using mouse tumor xenografts and focusing on the downregulation of PD-L1 (programmed death-ligand 1), a ligand of programmed cell death-1, a surface protein of activated T cells. Materials and Methods: B16/BL6 melanoma cells were injected into C57BL/6 or BALB/c-nu/nu mice to establish lung metastasis. BALB/c-nu/nu mice (nude mice) were used as a T cell–deficient model. The mice were given MK615 or saline orally every other day for approximately 8 weeks, and their survival was observed. NF-κB (nuclear factor-κB) and PD-L1 expressions of metastatic lung tissues were also examined. Results: The survival rate was improved only in the MK615-treated C57BL/6 mice ( P < .05), not in the saline-given control mice or BALB/c-nu/nu mice. The downregulations of NF-κB and PD-L1 were observed in both MK615-treated C57BL/6 and BALB/c-nu/nu mice. These results suggest that the antitumor effects of MK615 are associated with T cell–mediated immunity activated by MK-615-induced PD-L1 downregulation in tumor cells. Conclusion: MK615 is beneficial for a prolonged host survival time in the B16/BL6 melanoma xenograft model associated with T cell–mediated antitumor immunity.

Aim: The aim of this study was to identify gaps between Japan and the West in biomarker usage and the development of companion diagnostics. We also elaborated potential scenarios for companion diagnostic development. Methods: Information on drug labels in Japan, the USA and the EU was obtained from each regulatory authority's web site, as well as label information on in vitro diagnostic testing in Japan and the USA. Results: It is necessary to consider two factors when developing companion diagnostics globally: ethnic differences in gene mutations, and the approach to patient selection in clinical trials. A flowchart covering four scenarios was developed. Conclusion: Two factors that should be taken account when developing companion diagnostics globally was specified. This flowchart is expected to serve as a guide for streaming the development of companion diagnostics.

Cancer immunotherapy are taking a leading role of cancer therapy due to the development of the immune checkpoint blockade. To date, however, only about 20% of patients have clinical responses and the cancer-specific T cells in cancer site are required to obtain beneficial effects. There has been an innovative development in the field of adoptive cell therapy, especially receptor gene-modified T cells in recent years. The effector cells mostly express PD-1, therefore the cytotoxic reactivity of the effector cells are inhibited by PD-L1. The combination of the adoptive cell therapy and the immune checkpoint blockade is expected to enhance efficacy. On the other hand, the immune-related adverse events may also be enhanced, therefore, it is needed to develop the combination therapy carefully, improving the cancer antigen-specificity or dealing with the cytokine release syndrome.

Background The purpose of the present study was to explore novel biomarkers that can predict the clinical outcome of patients before treatment or during vaccination. These would be useful for the selection of appropriate patients who would be expected to exhibit better treatment outcomes from vaccination, and for facilitating the development of cancer vaccine treatments. Methods From a single-arm, non-randomized, human leukocyte antigen (HLA)-A-status-blind phase II trial of a vaccine treatment using three HLA-A*2402-restricted peptides for advanced pancreatic cancer (PC), we obtained peripheral blood samples from 36 patients of an HLA-A*2402-matched group and 27 patients of an HLA-A*2402-unmatched group. Results Multivariate analysis (HR = 2.546; 95% CI = 1.138 to 5.765; p = 0.0231) and log-rank test (p = 0.0036) showed that a high expression level of programmed death-1 (PD-1) on CD4+ T cells was a negative predictive biomarker of overall survival in the HLA-A*2402-matched group . Moreover, a high expression level of PD-1 on CD4+ T cells was a negative predictor for the induction of cytotoxic T lymphocytes (p = 0.0007). After treatment, we found that the upregulation of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) expression on CD4+ and CD8+ T cells was significantly associated with a poor clinical outcome in the HLA-A*2402-matched group (p = 0.0330, 0.0282, 0.0046, and 0.0068, respectively). In contrast, there was no significant difference for these factors in the HLA-A*2402-unmatched group. Conclusions Our results indicate that the upregulation of PD-1 and Tim-3 expression on CD4+ and CD8+ T cells may restrict T cell responses in advanced PC patients; therefore, combination immunotherapy with blockade of PD-1 and Tim-3 to restore T cell responses may be a potential therapeutic approach for advanced PC patients. Trial registration Clinical-Trail-Registration: UMIN000008082. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0509-1) contains supplementary material, which is available to authorized users.

Comprehensive genetic analysis of colorectal malignant tumors by microarrays has identified translocase of the outer mitochondrial membrane 34 (TOMM34) and ring finger protein 43 (RNF43) as highly expressed oncogenes in malignant colorectal tumors. Vaccine therapy using cancer peptides synthesized using the amino acid sequences of tumor antigens is currently undergoing clinical trials. Since it is important to perform vaccine therapy based on the oncogene expression levels in individual tumors, analysis of tumor antigen expression is necessary for this therapy. However, the quality of the messenger RNA extracted from formalin-fixed and paraffin-embedded specimens is generally considered insufficient for gene quantification. The present study examined whether it could be possible to quantify the expression of TOMM34 and RNF43 in colorectal cancer and liver metastasis samples prepared from paraffin blocks. The formalin-fixed and paraffin-embedded specimens were sliced for slides and the colorectal cancer and normal mucosal tissues were obtained from the slides. Total RNA was extracted from the tissue samples, and quantitative polymerase chain reaction (qPCR) was performed using the Universal ProbeLibrary as a PCR probe. Quantification of TOMM34 and RNF43 gene expression in several-year-old paraffin-embedded colorectal cancer specimens was possible by qPCR using the Universal ProbeLibrary. qPCR revealed that TOMM34 expression was elevated in 78.9% (15 of 19 cases) of the primary tumors and in 73.7% (14 of 19 cases) of the liver metastasis samples. RNF43 expression was elevated in 63.2% (12 of 19 cases) of the primary tumors and in 73.7% (14 of 19 cases) of the liver metastasis samples.

BACKGROUND: The standard treatment of resectable distal bile duct cancer is pancreatoduodenectomy. However, prognosis is still poor because of a high rate of lymph node (LN) metastasis, underlining the need to further clarify the mechanism of LN metastasis. Recently accumulated evidence has shown that cancer cells can take advantage of the homeostatic mechanism of lymphocyte homing to metastasize to LNs. The CXCL12-CXCR4 axis modulates lymphocyte homing by inducing continuous migration of dendritic cells and T cells that are expressing chemokine receptor CXCR4 to LNs where chemokine CXCL12 is homeostatically secreted. OBJECTIVE: The aim of this study is to elucidate whether distal bile duct cancer cells express CXCR4 and investigate a possible association with LN metastasis. METHODS: This study enrolled 32 patients with resectable distal bile duct cancer including 19 patients with LN metastasis and 13 without. All patients underwent pancreatoduodenectomy at our department between 2002 and 2008 and have been followed up (Median observation period: 50.5 (4-184) months). Histopathologically, 27 cases were tubular adenocarcinoma, and 5 cases were other types of cancer. Immunostaining with resected specimens was undertaken to examine the expression of CXCR4 in cancer cells and determine whether its expression level is associated with lymph node metastasis. When 10% and more of tumor cells were stained, specimens were diagnosed as positive for CXCR4. The result of staining level was visually assessed. RESULTS: Cancer cells were stained heterogeneously by antibody for CXCR4. Normal cells of the bile duct were not stained, while damaged or desquamated cells were stained. With regard to the staining pattern, the cytoplasm and membrane were stained but the nucleus was not stained. 19 out of 32 cases were positive for CXCR4 expression (59.4%). 15 of 19 cases with LN metastasis were positive whereas 4 of 13 cases without LN metastasis were positive (p=0.0107). CXCR4 expression on cancer cells increased with advancing cancer cell undifferentiation in tubular adenocarcinoma; cancer cells were positive for CXCR4 in 0 of 5 (0%), 6 of 16 (37.5%), and 6 of 6 (100%) cases in tubular 1, tubular 2, and tubular 3 adenocarcinoma, respectively. CONCLUSIONS: Our results indicated that the expression of CXCR4 in cancer cells may be associated with LN metastasis in distal bile duct cancer. Citation Format: Yohsuke Yagawa, Atsushi Aruga, Nobuhiro Takeshita, Ryota Higuchi, Takehisa Yazawa, Keishi Tanigawa, Masakazu Yamamoto. Association of CXCR4 expression with lymph node metastasis in distal cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1126.

Therapeutic cancer vaccines are an immunotherapy that targets tumor antigens to induce an active immune response. To date, Provenge® is the only therapeutic cancer vaccine approved by the United States Food and Drug Administration. Although therapeutic cancer vaccines have not been approved by the European Medicines Agency (EMA), they have been approved in several countries other than the United States (US) and the European Union (EU). Provenge® is the only approved cancer vaccine that showed significant primary endpoint efficacy in a phase III study at the time of approval. Retrospective analysis of 23 completed or terminated phase III studies showed that 74% (17/23) failed to demonstrate significant efficacy in the primary endpoint. The reasons for failure were surveyed in 13 of the 17 studies. Despite efforts to minimize tumor burden, including surgery and induction chemotherapy before therapeutic cancer vaccine therapy, 69% (9/13) of the phase III studies failed. These findings indicate that tumor burden may not be the only prognostic factor. Immunological response has often been used as a predictive factor, and a small number of sub-group analyses have succeeded in showing that immunological response is associated with the efficacy of therapeutic cancer vaccines. Being a prognostic factor, inclusion of immunological response in addition to tumor stage in the eligibility criteria or sub-group analysis may minimize study population heterogeneity, a key factor in the success of phase III studies.

Recently, analysis of tumor antigens using micro-arrays has revealed upregulation of cancer-testis antigens RNF43 and TOMM34 and vascular endothelial growth factor receptors VEGFR1 and VEGFR2 in colorectal cancer. A phase I clinical trial of peptide vaccine therapy together with oral anticancer drugs was conducted to treat advanced colorectal cancer using synthesized peptides of these tumor antigens in order to confirm the safety, immunogenicity and activity of this treatment. The subjects were patients with a human leukocyte antigen (HLA) type of A2402 who had inoperable colorectal cancer but had failed to respond to or were unable to undergo standard chemotherapy. Four peptides (RNF43, TOMM34, VEGFR1 and VEGFR2) were emulsified with incomplete Freund's adjuvant (Montanide), and the resulting solution was administered subcutaneously once a week. Patients received the oral anticancer drug tegafur-uracil plus leucovorin for four weeks continuously as part of one course followed by one week of rest. The primary endpoint of the trial was observation of adverse events as determined by the NCI-CTCAE criteria, and the secondary endpoints were the size of the tumor and the number of cytotoxic T lymphocytes (CTLs) in the peripheral blood after treatment. Vaccine therapy was administered 148 times to 10 patients from July 2008 to December 2009. The adverse events were grade 1 redness and induration, a grade 2 skin ulcer at the vaccination site and grade 1 pyrexia. All patients tolerated treatment. Tumor imaging revealed that after 1 course of treatment 1 patient had partial response (PR), 7 had stable disease (SD) and 2 had progressive disease. A CTL assay of 10 patients revealed an increase in peptide-specific CTLs in patients with PR and SD, and the clinical responses of those patients were observed. Kaplan‑Meier analysis indicated that patients who had a strong CTL reaction had a tendency to have longer progression‑free survival and overall survival.

Masaya Goto,1 Yoshihiro Muragaki,2 Atsushi Aruga1 1Cooperative Major in Advanced Biomedical Sciences, Joint Graduate School of Tokyo Women's Medical University and Waseda University, 2Intelligent Clinical Research and Innovation Center, Tokyo Women's Medical University, Tokyo, Japan Abstract: In investigator-initiated clinical trials, protocols with inappropriate methods might cause bias. However, insufficient data are available to determine which items are important or difficult to discuss in protocol development. We recorded protocol-writing support conferences to determine what items methodologists and investigators discussed. We obtained approval from all applicants to attend our Intelligent Clinical Research and Innovation Center writing support conferences, recorded all the discussions, characterized them, and sorted the items iteratively. In 1 year, we had 18 conferences: nine early protocol conferences and nine rejected protocol conferences. The latter were rejected by the institutional review board, which requested consultation. The most discussed item was outcomes, accounting for ~20% of the total discussion time. In three trials, the main problem was multiple primary outcomes. The second most discussed item was control. Early protocol conferences had more non-preliminary proposal items than rejected ones (P

Adoptive cell therapy using autologous αβ-T cells is one of the most effective treatments for various cancers and has minimal side effects. αβ-T cells have the ability to recognize the cancer antigens that present with human leukocyte antigen (HLA) molecules on the cancer cells and kill them by secreting several granules, such as perforin, granzyme, or granulysin. Several clinical effects have been defined in clinical trials, but standardized treatment approaches have not been established. Because of the difficulty of dealing with several regulations and the high costs, there have been very few large clinical trials. Even so, recent technical advances in such fields as genetic engineering may make it possible to cure various cancers using new candidates for αβ-T-cell therapy. It is expected that safe and effective αβ-T-cell therapy will be developed as a standard cancer therapy in the near future.

We have already reported the extremely high thermoelectric properties of Si–Ge–Au amorphous thin films. The thermoelectric properties of such films change slowly during annealing cycles. In this paper, we describe the absorption spectra of the films by using photoacoustic spectroscopy (PAS) to clarify the change mechanism of these thermoelectric properties. Si–Ge–Au amorphous thin films were prepared by the alternate deposition of a non doped Si layer and a heavily Au doped Ge layer. The peak positions of the absorption spectra shift in a complex way with annealing, where the thermoelectric properties change. In these annealing cycles, we assume three stages to explain the mechanism of the change in the thermoelectric properties: the collapse of the superlattice, compositional diffusion and the termination of recrystallization at 673 K. We conclude that the change in the thermoelectric properties is explained by these three stages.

Ag-doped Cu2SiS3 particles were prepared by the reaction of elements at 800 °C for 7 days. Their crystal structure were determined from single crystal X-ray diffraction data. The solid solution limit of Ag atom into Cu atom in Cu2SiS3 was estimated to be 2.0 mol % from the change of unit cell volumes. Crystal structure of 2 mol % Ag-doped Cu2SiS3 was isostructure with monoclinic Cu2SiS3, superstructure of 3 times of sphalerite type. Optical absorption of above particles, whose Ag contents were from 0.0 to 3.0 mol % against to Cu, was measured by a microphone photoacoustic spectroscopy. Midpoint of main transition on Cu2SiS3 particles was 499 nm (2.48 eV) and an absorption in the IR region was from 710 to 730 cm-1 corresponding to the activation energy of Cu atom bonded by S atoms. The effect of Ag addition was almost nothing up to the limit of solid solution of 2 mol % Ag, but excess Ag addition caused to a slight shift to a wide gap. So it might be consider to a photoelectric or photocatalytic application by use of sunlight.

The luminescence properties of Eu 2− x Y x W 2 O 9 (0 ≦ x ≦ 1.8) were studied. These samples were prepared by solid-state reaction. The purpose of this work was to see the luminescence variations due to the amount of Y 3+ ions and preparation temperatures. The samples 0–50 mol% Y 3+ -doped and fired at 900–1100 °C showed a single phase. In them, 20 mol% Y 3+ -doped and fired at 1100 °C showed high emission intensity at the range of 614 nm. On the other hand, 30 mol% Y 3+ -doped samples fired at 1150 and 1200 °C included Eu 2 W 3 O 12 as another component. The highest luminescence intensity was shown in 30 mol% Y 3+ -doped and fired at 1150 °C. It seems that another component related to the high luminescence intensity in these phosphors.

Therapeutic cancer vaccines are an immunotherapy that amplify or induce an active immune response against tumors. Notably, limitations in the methodology for existing anti-cancer drugs may subsist while applying them to cancer vaccine therapy. A retrospective analysis was performed using information obtained from ClinicalTrials.gov, PubMed, and published articles. Our research evaluated the optimal methodologies for therapeutic cancer vaccines based on (1) patient populations, (2) immune monitoring, (3) tumor response evaluation, and (4) supplementary therapies. Failure to optimize these methodologies at an early phase may impact development at later stages; thus, we have proposed some points to be considered during the early phase. Moreover, we compared our proposal with the guidance for industry issued by the US Food and Drug Administration in October 2011 entitled “Clinical Considerations for Therapeutic Cancer Vaccines”. Consequently, while our research was aligned with the guidance, we hope it provides further insights in order to predict the risks and benefits and facilitate decisions for a new technology. We identified the following points for consideration: (1) include in the selection criteria the immunological stage with a prognostic value, which is as important as the tumor stage; (2) select immunological assays such as phenotype analysis of lymphocytes, based on their features and standardize assay methods; (3) utilize optimal response criteria for immunotherapy in therapeutic cancer vaccine trials; and (4) consider supplementary therapies, including immune checkpoint inhibitors, for future therapeutic cancer vaccines.

Gene-based vaccines as typified by plasmid DNA vaccines and recombinant viral-vectored vaccines are expected as promising solutions against infectious diseases for which no effective prophylactic vaccines exist such as HIV, dengue virus, Ebola virus and malaria, and for which more improved vaccines are needed such as tuberculosis and influenza virus. Although many preclinical and clinical trials have been conducted to date, no DNA vaccines or recombinant viral-vectored vaccines expressing heterologous antigens for human use have yet been licensed in the U.S., Europe or Japan. In this research, we describe the current regulatory context for gene-based prophylactic vaccines against infectious disease in the U.S., Europe, and Japan. We identify the important considerations, in particular, on the preclinical assessments that would allow these vaccines to proceed to clinical trials, and the differences on the regulatory pathway for the marketing authorization in each region.

Aim: This study aimed to derive meaningful parameters for immune monitoring during cancer vaccine development by analysis of the literature. Methods: This retrospective study was based on analysis of clinical trials registered at ClinicalTrials.gov and published data available on PubMed. Results: The most common sample evaluated in immune monitoring was peripheral blood. All trials employed ELISA for detecting a humoral immune response; however, cellular immune assays were not used across trials. Most cellular immune assays failed to correlate with clinical outcome, although results of other methods did. Conclusion: Standardization of the cellular immune assays across trials is important for predicting the effects of therapeutic cancer vaccines when considering the reliability and characteristics of the methods. Currently, assays mostly target detection of T-cell function, such as proliferation and cytokine release; however, T-cell phenotype analysis in peripheral blood and/or tumor sites may also be considered in the future.

繊維含有量44vol%の三次元炭素繊維強化フラン樹脂 (3D-CFRFR) を作製し, 1000℃以下の種々の温度で加熱した. 1000℃の加熱による体積収縮は, フラン樹脂単体で50%であったが, 3D-CFRFRでは数%であった. 曲げ強さは加熱処理温度 (HTT) の上昇に伴って減少した. フラン樹脂に対する繊維強化効果はHTT500℃以下で認められた. 加熱に伴う曲げ強さの低下は, マトリックスと炭素繊維境界面のはく離と炭素繊維の損傷による. はく離や損傷はマトリックスのフラン樹脂の熱分解を通して起こる. 炭素繊維積層表面直角方向 (σs, Es) の曲げ強さと曲げ弾性率は, 厚さ直角方向 (σt, Et) のそれらより小さかった. 両方向の曲げ強さおよび両方向の曲げ弾性率には直線関係があり, それぞれσt=2.7σs, Et=1.7Esで表される.

Background and Objectives The sensitivity and specificity of the monoclonal antibody Hepatocyte Paraffin 1 (Hep Par 1) for hepatocellular carcinoma (HCC) are very high, and the usefulness for differential diagnosis of hepatic tumors has been reported. However, there are some cases of HCC with negative staining for Hep Par 1. We examined the histopathological features of HCC with negative staining for Hep Par 1. Methods We examined 69 samples of single nodular HCC less than 2 cm in greatest dimension, resected from 1985 to 1994 in our hospital, with immunohistological staining for Hep Par 1, cytokeratin 19 (CK 19), MUC-1 glycoprotein (MUC-1), and epithelial membrane antigen (EMA). Results Hep Par 1 staining was positive in 64 cases (93%) and negative in 5 cases (7%). With regard to the histological structure, 3 of the 5 negative cases were scirrhous HCC. With regard to the grade of histological differentiation, 2 cases were poorly differentiated HCC, 3 cases were moderately differentiated HCC, and no well-differentiated HCC was found in the negative cases. CK 19, MUC-1, and EMA staining were negative in all cases. Conclusions It is necessary to recognize the existence of Hep Par 1 negative HCC, in particular scirrhous HCC. This may be due to a different mechanism in the earlier stage of hepatocarcinogenesis. J. Surg. Oncol. 2004;88:104–107. © 2004 Wiley-Liss, Inc.

BACKGROUND It has been reported that MUC1 is an important prognostic factor in several cancers. This study investigated the importance of MUC1 as a prognostic factor in mass-forming intrahepatic cholangiocarcinoma (m-ICC). METHODS In 50 patients with m-ICC who had undergone hepatectomy, expression of MUC1 was investigated. Expression of MUC1 was examined by immunohistochemical staining with monoclonal antibody HMPV, which recognizes the MUC1 core peptide. The immunohistochemical staining patterns of MUC1 were classified into three types: ductal type (the luminal surface membrane of neoplastic cells was stained), cytoplasmic type (the cytoplasm of neoplastic cells was stained dominantly), and negative type. RESULTS Expression of MUC1 was detected immunohistochemically in 38 (76%) of 50 cases of m-ICC (ductal type, 18; cytoplasmic type, 20; and negative type, 12). Seventy-five percent of patients with lymph node metastasis had the cytoplasmic type MUC1 expression. Lymph node dissection was performed in only 20 patients, but significant correlation was demonstrated between MUC1 expression and lymph node metastasis (P = 0.0227). The location of MUC1 expression correlated with surgical outcome in m-ICC. Patients with the cytoplasmic type expression showed significantly lower survival rates. Univariate analysis revealed that MUC1 expression was a statistically significant risk factor affecting outcome in m-ICC (P = 0.0028). Furthermore, expression of MUC1 was found to be a statistically significant independent risk factor in multivariate analysis (P = 0.0063). CONCLUSIONS The results suggest that evaluation of MUC1 expression may be very useful in predicting the surgical outcome in m-ICC. Cancer 2002;94:1770–6. © 2002 American Cancer Society. DOI 10.1002/cncr.10398

Standard in vivo cancer models entail injecting single cancer cells, but this technique is not always successful. We developed a novel cancer cell sheet by using temperature-responsive polymer poly(N-isopropyl acryl amide)-coated plates, which allow controlled attachment and detachment of living cancer cells via simple temperature changes.Four human cancer cell lines were used to make cell sheets. The cancer cell sheets were subcutaneously transplanted into nude mice and compared regarding their tumor-forming ability with the conventional cell suspension technique.Human cancer cell sheets were successfully transplanted into nude mice. The cancer cell sheets resulted in stable engraftment and showed a higher tumor volume determined by total flux with the IVIS® imaging system.Novel cancer cell sheets are useful tools to make in vivo cancer models in mice for the assessment of anticancer therapeutics.

Increase in body temperature has been thought to play an important role in the regulation of immune responses, although its precise mechanisms are still under investigation. Here, we examined the effects of physiologically relevant thermal stress on the cytokine production from human peripheral T cells. Volunteers were heated using a whole-body hyperthermia device, the rectal temperature was maintained above 38.5 °C for more than 60 min, and peripheral blood mononuclear cells (PBMCs) were obtained before and after the treatment. When T cells were stimulated with anti-CD3/CD28 antibodies, marked increases in the production of interferon-γ (IFN-γ) and interleukin-2 were observed in PBMCs prepared immediately after and 24h after the treatment. Similarly, enhanced production of IFN-γ in response to the tuberculin purified protein derivative or antigenic viral peptides was also observed immediately after and 24h after the treatment. Fluorescence photo-bleaching analyses showed heat-induced increase of membrane fluidity in T cells, which probably enables them to induce rapid and efficient cluster formation of molecules involved in antigen recognition and signal transduction for T-cell stimulation. We concluded that physiologically relevant thermal stress could efficiently modify T-cell responsiveness to various stimuli, including enhanced responses to specific antigens.

Recently, studies on regenerative stem cell therapy are being encouraged, and efforts to generate dendritic cells, which play important roles in cancer immunotherapy, from stem cells are being made in the field of tumor immunology. Therapeutic acquisition of stem cells has important clinical applications. Studies on induced pluripotent stem(iPS)cells generated from somatic cells with pluripotent genes have advanced in recent years. Stem cells are reported to be found in adipose tissue (adipose-derived stem cells, ADSC). Our goal is to develop a new cancer vaccine by using dendritic cells generated from ADSC. In a preliminary study, we examined whether iPS cells can be generated from ADSC to serve as a source of dendritic cells.We introduced a plasmid with pluripotent genes(OCT3/4, KLF4, SOX2, L-MYC, LIN28, p53-shRNA)into an ADSC strain derived from adipose tissue by electroporation and subsequently cultured the cells for further examination. A colony sugges- tive of iPS cells from ADSC was observed. OCT3/4, KLF4, SOX2, L-MYC, and LIN28 mRNAs were expressed in the cultured cells, as confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR). On the basis of these results, we confirmed that iPS cells were generated from ADSC. The method of inducing dendritic cells from iPS cells has already been reported, and the results of this study suggest that ADSC is a potential source of dendritic cells.

The recurrence rate after surgery in patients with hepatocellular carcinoma (HCC) is very high, while prognosis is quite poor. However, there is no standard treatment to prevent recurrence of HCC after a curative operation. In this study, we investigated the clinical utilization of an autologous tumor lysate-pulsed dendritic cell vaccine plus ex vivo activated T cell transfer (ATVAC) in an adjuvant setting for postoperative HCC as a non-randomized controlled trial. Ninety-four patients with invasive HCC received informed consent information regarding the study, and 42 opted to have the ATVAC after surgery. Their recurrence-free survival (RFS) and overall survival (OS) were measured after 5 years and compared with those of 52 patients who selected to have the curative operation alone. The median RFS and OS were 24.5 months and 97.7 months in the patients receiving adjuvant ATVAC and 12.6 months and 41.0 months in the group receiving surgery alone (P = 0.011 and 0.029). In the treated group, patients with positive delayed-type hypersensitivity (DTH) had a better prognosis (RFS P = 0.019, OS P = 0.025). No adverse events of grade 3 or more were observed. A postoperative dendritic cell vaccine plus activated T cell transfer would be a feasible and effective treatment for preventing recurrence in HCC patients and achieving long-term survival especially in DTH positive patients.

ケブラークロスとフルフリルアルコール樹脂(フラン樹脂)から作製した積層複合材料の熱処理に伴う強度変化を，600 °C以下の温度で熱処理した試料片の曲げ試験から明らかにした．積層複合材料の繊維含有量は39 vol%で，マトリックスのフラン樹脂とほぼ同等の曲げ強度を示した．熱処理温度200–500 °Cの間で繊維強化効果が認められた．クロス積層複合材料の特徴は，荷重 vs. たわみ曲線に現れた．すなわち，Fig. 8cに示したように，ある荷重下で突然の曲げ破壊は起こらなかった．しかし，炭素化が進行する500 °C以上の熱処理試料ではこの特性は消失した．熱処理試料の曲げ強度(σ)とかさ密度(ρ)には，σ ∝ ρn の関係が認められた．さらに，クロス積層面に平行方向について，曲げ弾性率対曲げ強度のプロットをとると直線関係となったが，垂直方向では直線関係とならなかった．

The Fukushima nuclear accident has highlighted the importance of finding a better final storage method for radioactive cesium species. Cs is highly soluble in water, and can easily exchange with other alkali ions in zeolites or clays to form stable complexes. However, Cs(+) is released from Cs(+) complexes into water when surrounded by an excess of water. Pollucite may be the best final storage option for Cs(+), but its typical synthesis requires heating to about 1200 °C in air. Here, we show that the hydrothermal synthesis of pollucite can be completed at 300 °C in three hours from any zeolite or clay. Furthermore, our procedure does not require ion exchange before synthesis. Radioactive Cs is usually found in complexes with clays. At that time, this method only requires calcium hydroxide, water, and three hours of hydrothermal synthesis, so the process is both inexpensive and practical for large-scale application. Pollucite is an analog of analcime zeolite, and contains a channel system 2.8 A in diameter, which is formed by 6-oxygen rings. As the diameter of Cs(+) is 3.34 A and each Cs(+) exists independently within a separate portion of the channel, Cs(+) cannot exit the pollucite framework without breaking it.

Meeting abstracts Previous studies in mice have showed that fever-range thermal stress (approx. 38.5-40.5 degrees C) stimulates a variety of T cell functions ex vivo and in vivo. The aim of this study is to examine the effects of increase in core body temperature in human system on the function of