Akimune Hayashi

Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-γ

We investigated the feasibility of local treatment or tumor vaccination with a herpes simplex virus (HSV) type 1-defective vector. The vector was engineered to express murine interferon-γ (IFN-γ) for experimental gene therapy against mouse glioma Rous sarcoma virus (RSV). The murine IFN-γ gene was driven by the cytomegalovirus promoter. The helper virus (tsk) was thermosensitive; consequently, this vector could only proliferate at 31°C. A high level of murine IFN-γ expression was confirmed in vitro and in vivo by immunohistochemistry using anti-mouse IFN-γ monoclonal antibody. This engineered vector (dvHSV/MuIFN-γ) inhibited the proliferation of mouse glioma RSV cells in vitro, and an intratumoral (i.t.) local injection of the vector caused i.t. necrosis in vivo. The immunological effect of dvHSV/MuIFN-γ was also examined in a mouse glioma RSV cell implantation model. A subcutaneous (s.c.) implant of 1 × 106 mouse glioma RSV cells after treatment with dvHSV/MuIFN-γ was rejected. However, the implant after treatment with an engineered HSV-defective vector containing an antisense nucleotide sequence of the murine IFN-γ gene was not rejected. In addition, in another group of mice in which RSV cells treated with dvHSV/MuIFN-γ were implanted into a femoral (s.c.) region and nontreated RSV cells were implanted into a contralateral femoral (s.c.) region, the implanted RSV cells were rejected. The rejection of the implanted mouse glioma RSV was blocked by anti-asialo GM1, which was known to inhibit natural killer cell activity. These results revealed that the HSV-defective vector could realize a high efficiency of transfection to glioma cells through short-time treatment, and that the IFN-γ gene transferred to the cells had the effect of tumor vaccination, which was suggested be related to natural killer cells. In conclusion, dvHSV/MuIFN-γ may be useful for the gene therapy of malignant glioma through either i.t. local injection or a practical tumor vaccination with ex vivo gene transfer.

Cosmetic reconstruction after mastoidectomy for the transpetrosal-presigmoid approach : Technical note

OBJECTIVE We describe the technique of cosmetic reconstruction after mastoidectomy in the transpetrosal-presigmoid approach for petroclival lesions. TECHNIQUES This technique involves raising a single temporo-occipital bone flap and cosmetic mastoidectomy, removing the mastoid bone fragments (by means of a rongeur instead of an air drill) for later reconstruction. Replacement of the bone fragments mixed with fibrin glue enables tamponade against the dura to be achieved without a fat graft. RESULTS Computed tomographic scans and plain cranial films taken a few years later showed successful reconstruction of the mastoid bone with a good appearance of the retromastoid area in seven patients who underwent this procedure. The only complication was infection in one patient from cerebrospinal fluid leakage from the wound. CONCLUSION We have developed a simple and easy technique of cosmetic reconstruction after the transpetrosal-presigmoid approach.

Establishment of patient-derived cancer xenografts in immunodeficient NOG mice

Viable and stable human cancer cell lines and animal models combined with adequate clinical information are essential for future advances in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available; however, they lack detailed information on the patient from which they originate, including disease phenotype and drug sensitivity. Patient-derived xenografts (PDX) with clinical information (so-called ‘cancer xenopatients’) are a promising advance that may accelerate the development of anticancer therapies. We established 61 PDX lines from 116 surgically removed tumor tissues inoculated subcutaneously into NOG mice (53% success rate). PDX lines were established from various types of epithelial tumors and also from sarcomas, including gastrointestinal stromal tumors and Ewing/PNET sarcomas. The metastatic tumors yielded PDX lines more effectively (65%) than the primary tumors (27%, P<0.001). In our PDX models, morphological characteristics, gene expression profiles, and genetic alteration patterns were all well preserved. In eight cases (7%), the transplantable xenografts for several generations were composed of large monotonous nonepithelial cells of human origin, revealed to be Epstein-Barr virus infection-associated lymphoproliferative lesions. Despite this, PDX linked with clinical information offer many advantages for preclinical studies investigating new anticancer drugs. The fast and efficient establishment of individual PDX may also contribute to future personalized anticancer therapies.

Collagen gel matrix assay as an in vitro chemosensitivity test for malignant astrocytic tumors

BackgroundThe efficacy of individual chemotherapy based on chemosensitivity has scarcely been studied.MethodsWe examined the chemosensitivites for four anticancer agents – 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3 (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), carboplatin, cisplatin, and etoposide – of 43 malignant astrocytic tumors (21 anaplastic astrocytomas and 22 glioblastomas) by using a collagen gel matrix assay, and we also determined the survival periods of the tumor-bearing patients. The chemosensitivity was evaluated in terms of the growth inhibition rate, using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) method.ResultsFor the anaplastic astrocytomas, the mean growth inhibitory rate was 33.2% with cisplatin, 37.2% with carboplatin, 28.0% with ACNU, and 24.8% with etoposide. For the glioblastomas, these rates were 36.9%, 42.3%, 23.2%, and 34.8%, respectively. The median overall and progression-free survivals of anaplastic astrocytoma-bearing patients who had undergone chemotherapy with two anticancer drugs, both of which showed significant anticancer activity (growth inhibitory rate >30%) were significantly longer than those of the patients who had been treated with two drugs, one or both of which did not show significant anticancer activity. On the other hand, there was no significant difference in the overall or the progression-free survivals in the two corresponding groups of glioblastoma-bearing patients.ConclusionThe collagen gel matrix assay is clinically useful to determine in vitro chemosensitivity that reflects in vivo chemosensitivity. Individual chemotherapy for malignant astrocytic tumors, based on chemosensitivity data, could contribute to longer survival, particularly in anaplastic astrocytoma-bearing patients.

Abstract B38: Clinical applications of PDX/NOG models for personalized chemotherapy – possible use in chemo-sensitivity testing and clinical sequencing

Personalized medicine represents an ideal medical approach for cancer therapy. In the field of clinical oncology, personalized medicine or therapy involves the evolutionary expansion of conventional clinical approaches that progress from patient evaluation, differential diagnosis, to the treatment of diseases. A number of the complex techniques employed in personalized medicine, such as clinical genome sequencing, biochemical marker analyses, chemo-sensitivity testing, and cancer immunotherapy, require fresh, viable, and sufficient amounts of specimens for reliable estimations. Mice bearing patient-derived xenografts (PDXs) with clinical information (so-called “Cancer Xenopatients”) are remarkable systems in personalized medicine for cancer. We previously reported the rapid and efficient establishment of PDXs using NOG mice (PDX/NOG models, AACR2015 #1940, IJO 47 61-70 2015). NOG mice, NOD/Shi-scid/IL2Rγ null (NOG) mice derived from NOD/SCID mice with a common gamma chain, have multifunctional defects in natural killer cell activity, macrophage function, complement activity, and dendritic cell function in addition to the absence of functional T and B lymphocytes. NOG mice have been identified as the most appropriate immunodeficient host animal for the direct xenografting of fresh tumor tissue due to the preservation of cancer stem cells (CSCs). Fresh and valuable xenograft samples, similar to surgical samples with the preservation of CSC, are stably provided using PDX/NOG models. Moreover, human tissue (tumor) and mouse tissue (stroma) are clearly distinguished by immunohistochemical analysis or gene arrangement sequencing. In the present study, we discussed the possibility of using PDX/NOG model simulations for personalized cancer chemotherapy. We previously established 47 lines of gastrointestinal cancer xenografts. In these cases, clinical information regarding chemotherapy for donor patients was retrieved where possible. Collagen gel droplet-embedded culture-drug sensitivity tests (CD-DST) were performed on 16 lines of PDX/NOG. In 4 of these lines, CD-DST were successfully conducted on original surgical specimens. The results of CD-DST between original and PDX/NOG specimens generally correlated (R 2 =0.01-0.89). The results of CD-DST using PDX/NOG specimens were compatible with the clinical effects of anti-cancer drugs. Genome sequencing and interactome analyses, a comprehensive analysis of tumor-stroma interactions innovated by Professor Ishikawa S. at Tokyo Medical and Dental University, were also performed on mRNA from 17 lines of PDX/NOG. Our interactome analyses showed tumor-stroma interactions in PDX/NOG comprehensively and quantitatively at the gene-expression level by distinguishing gene arrangements in human tissue (tumor) from mouse tissue (stroma). The EGF-EGFR or VEGFA-KDR interactions observed closely reflected the clinical effectiveness of an EGFR inhibitor (Cetuximab) or VEGF-A inhibitor (Bevacizumab) as well as the results of in vivo chemo-sensitivity tests using PDX/NOG. The results of CD-DST and sequencing in PDX/NOG appear to be reliable for clinical simulations of chemotherapy and will definitely assist in the selection of the most sensitive anti-cancer drug for each patient. The fast and efficient establishment of individual PDXs will contribute to personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Takayuki Isagawa, Akira Noguchi, Hidemitsu Sato, Akimune Hayashi, Haruhiko Cho, Manabu Shiozawa, Takeshi Kishida, Soichiro Morinaga, Tomoyuki Yokose, Makoto Katayama, Nobuo Takenaka, Mizuha Haraguchi, Naoki Miyao, Yuichi Tateishi, Kenji Kawai, Hiroshi Suemizu, Roppei Yamada, Yoshiyasu Nakamura, Kohzoh Imai, Daisuke Komura, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. Clinical applications of PDX/NOG models for personalized chemotherapy – possible use in chemo-sensitivity testing and clinical sequencing. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B38.

Abstract 1449: The valuable experimental model system “Cancer Xenopatient” established in NOG mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Viable and stable human cancer materials combined with adequate clinical information are required for advance in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available without enough information on the patient including disease phenotypes and drug-sensitivity. Mice bearing Patient-derived xenografts (PDXs) with clinical information (so-called “cancer xenopatients”) could be essential and useful systems to accelerate cancer medicine. In this study, we investigated the establishment efficiency of PDX using NOG mice with clinical factors of xeno-transplantaion. The NOG mouse, the NOD/Shi-scid/IL-2Rγnull (NOG) mice derived from the NOD/SCID mouse with a common gamma chain, has multifunctional defects in natural killer cell activity, macrophage function, complement activity, and dendritic cell function in addition to lacking functional T and B lymphocytes. NOG mice were reported to be the best appropriate immunodeficient host animal for direct xenografting of fresh tumor tissue. We also discuss herein its contribution for not only reliable preclinical studies of new anticancer drugs but also personalized anti-cancer therapies. Sixty-one PDX lines were established from 116 surgically removed tumor tissues inoculated into NOG mices subcutaneous (53%). PDX lines were established from various types of epithelial tumors and also from sarcomas including gastrointestinal stromal tumor and Ewing/PNET sarcoma. The metastatic tumors yielded PDX lines effectively (65%) than the primary tumors (27%, p<0.001). The group engrafted into NOG mice after 2 days or later from the surgical removal showed a higher establishment rate (61%) than that of the group engrafted at the day of surgery or the next day (51%), however non-significant (p = 0.49). Our PDX models were preserved well not only in morphological characteristics but also in gene expression and alteration patterns. In 8 cases (7%), the transplantable xenografts for several generations were composed of monotonous large non-epithelial cell growth of human origin, revealed to be the Epstein-Barr virus (EBV) infection associated lymphoprolipherative lesions prospected by chromogenic in situ hybridization for EBV-encoded RNA. We revealed efficient establishment rates both for primary tumors and metastatic tumors. PDXs linked with clinical information will contribute to reliable preclinical studies for new anticancer drugs. The fast and efficient establishment of individual PDXs may also contribute to future personalized anti-cancer therapies. Citation Format: Tsuyoshi Chijiwa, Kenji Kawai, Akira Noguchi, Hidemitsu Sato, Akimune Hayashi, Haruhiko Cho, Manabu Shiozawa, Takeshi Kishida, Soichiro Morinaga, Tomoyuki Yokose, Makoto Katayama, Nobuo Takenaka, Hiroshi Suemizu, Roppei Yamada, Yoshiyasu Nakamura, Yasuo Takano, Kohzoh Imai, Yohei Miyagi, Masato Nakamura. The valuable experimental model system “Cancer Xenopatient” established in NOG mice. [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 1449. doi:10.1158/1538-7445.AM2015-1449