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Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. High-risk NB is characterized by MYCN amplification and human telomerase reverse transcriptase (hTERT) rearrangement, contributing to hTERT activation and a poor outcome. For targeting hTERT-activated tumors, we developed two oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in which the hTERT promoter drives expression of the viral E1 gene for tumor-specific virus replication. In this study, we demonstrate the therapeutic potential of the hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 using four human MYCN-amplified NB cell lines (IMR-32, CHP-134, NB-1, LA-N-5) exhibiting high hTERT expression. OBP-301 and OBP-702 exhibited a strong antitumor effect in association with autophagy in NB cells. Virus-mediated activation of E2F1 protein suppressed MYCN expression. OBP-301 and OBP-702 significantly suppressed the growth of subcutaneous CHP-134 tumors. Thus, these hTERT-driven oncolytic adenoviruses are promising antitumor agents for eliminating MYCN-amplified NB cells via E2F1-mediated suppression of MYCN protein., Graphical Abstract, MYCN-amplified neuroblastoma (NB) cells exhibit transcriptional activation of human telomerase reverse transcriptase (hTERT) expression. hTERT promoter-driven oncolytic adenoviruses OBP-301 and OBP-702 exhibited a strong antitumor effect in human MYCN-amplified NB cells via induction of autophagy and E2F1-mediated MYCN suppression.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Recent whole-genome sequencing studies have demonstrated that high-risk NB with unfavorable clinical outcome is characterized by amplification of MYCN oncogene and rearrangement of human telomerase reverse transcriptase (hTERT) gene. Since hTERT expression is transcriptionally and commonly activated in high-risk NB, hTERT activation would be an attractive therapeutic target for the treatment of high-risk NB. To eliminate hTERT-activated malignant tumor cells, we have developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702. In this study, we investigated the therapeutic potential of hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 in MYCN-amplified NB cells. Methods: We used 4 human NB cell lines with MYCN amplification (IMR-32, CHP-134, NB-1, LA-N-5). The expression of cell surface coxsackie and adenovirus receptor (CAR) and hTERT was analyzed using flow cytometric analysis and real-time RT-PCR. We assessed the in vitro antitumor effect of OBP-301 and OBP-702 using a XTT assay. The molecular mechanism of virus-mediated antitumor effect was investigated in aspect of apoptosis, autophagy, and MYCN expression. To address the underlying mechanism of virus-mediated MYCN modulation, we analyzed the role of E2F1 and p53 in the virus-infected NB cells. The in vivo antitumor effect of OBP-301 and OBP-702 was assessed using a subcutaneous CHP-134 xenograft tumor model. Results: All MYCN-amplified NB cells showed CAR and hTERT expression. OBP-301 and OBP-702 showed profound antitumor effect in association with autophagy-related cell death in all MYCN-amplified NB cells. In the virus-infected NB cells, E2F1 expression was inversely correlated with MYCN expression. Virus-induced E2F1 and p53 expression suppressed the MYCN expression in the MYCN-amplified NB cells. OBP-301 and OBP-702 significantly suppressed tumor growth in a subcutaneous CHP-134 xenograft tumor model. Conclusion: Our results suggest that hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 are promising antitumor agents for eliminating the MYCN-amplified NB cells through E2F1- and p53-mediated suppression of oncogenic MYCN protein. Further clinical study is warranted to evaluate the safety and feasibility of hTERT-driven oncolytic virotherapy as novel precision medicine for high-risk NB patients. Citation Format: Morimichi Tani, Hiroshi Tazawa, Terutaka Tanimoto, Hiroshi Nouso, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Elimination of MYCN-amplified neuroblastoma cells by telomerase-targeted oncolytic virotherapy as novel precision medicine [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 2873.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. High-risk NB patients show an unfavorable clinical outcome, even after treatment with intensive chemotherapy and aggressive surgical resection. Therefore, a novel therapeutic strategy is needed for the treatment of high-risk NB patients. We developed a tumor-specific replication-competent oncolytic adenovirus OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces p53 expression. Preclinical studies demonstrated that OBP-702 induces autophagy-related cell death in human NB cell lines. To enhance OBP-702-induced autophagy-related cell death, we hypothesized that a mTOR inhibitor temsirolimus, which is well-known autophagy inducer, may promote autophagy-related cell death by OBP-702 infection. In this study, we investigated the antitumor effect of OBP-702 in combination with temsirolimus in human NB cell lines. Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The antitumor effect of OBP-702 and temsirolimus was evaluated using XTT assay. In vitro synergistic effect was calculated with CalcuSyn software (BioSoft, Inc.). Virus-mediated cell death and p53 expression were analyzed by Western blot analysis. Ad-p53, a replication-defective adenovirus expressing p53 gene, was used to assess the effect of p53 in combination with temsirolimus. The combined effect of OBP-702 and temsirolimus was further evaluated using a subcutaneous CHP-134 xenograft tumor model. Results: Combination with OBP-702 and temsirolimus exhibited a synergistic antitumor effect in all NB cell lines in vitro. We confirmed that combination with these drugs enhanced autophagy. In addition, temsirolimus showed an effect to stabilize p53 protein induced by OBP-702. Ad-p53 also showed synergistic effect in combination with temsirolimus. The combination treatment significantly inhibited tumor growth, as compared to monotherapy, in NB xenograft tumor model. Conclusions: These results suggest that p53-expressing oncolytic adenovirus and mTOR inhibitor synergistically induce autophagic cell death and may be a promising strategy in NB patients. Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Morimichi Tani, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. mTOR inhibitor augments the antitumor effect of p53-expressing oncolytic adenovirus in human neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3177.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Despite the recent advances in the treatment of NB, patients with high-risk NB still have poor prognosis. Immunotherapy using anti-GD2 monoclonal antibody (mAb), which binds to disialoganglioside GD2 on the surface of NB cells, has recently become a standard treatment for high-risk NB. However, the therapeutic efficacy of anti-GD2 mAb is insufficient in many clinical trials. Therefore, the enhancement of therapeutic potential of anti-GD2 mAb is needed to improve the clinical outcome of patients with high-risk NB. Near-infrared photoimmunotherapy (NIR-PIT) is a promising antitumor strategy to enhance the therapeutic potential of immunotherapy using an antibody-photoabsorber conjugate (APC). In this study, we investigated the therapeutic efficacy of anti-GD2-based NIR-PIT using anti-GD2 mAb conjugated to the photoabsorber IR700 (anti-GD2-IR700) in human NB cells. Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The expression of GD2 on the cell surface of NB cells was analyzed by flow cytometric analysis. Anti-GD2-IR700 was synthesized by incubating anti-GD2 mAb and IR700. The mixture was purified with a Sephadex G50 column. The antitumor effect of anti-GD2-IR700 was evaluated using XTT assay. NB cells were incubated with anti-GD2-IR700, anti-GD2 mAb or PBS and NIR light was irradiated at 2.0J/cm2. Results: IMR-32 and CHP-134 cells, but not SK-N-SH cells, expressed GD2 protein on the cell surface. In GD2-positive IMR-32 and CHP-134 cells, administration of anti-GD2-IR700 significantly suppressed the cell viability compared to anti-GD2 mAb or PBS when combined with NIR light irradiation. In contrast, in GD2-negative SK-N-SH cells, anti-GD2-IR700 showed no significant inhibition of cell viability in combination with NIR light irradiation. Conclusion: These results suggest that NIR-PIT using an anti-GD2-IR700 is a promising antitumor strategy to promote the therapeutic efficacy of anti-GD2 immunotherapy for high-risk NB. Citation Format: Hiroshi Nouso, Hiroshi Tazawa, Terutaka Tanimoto, Morimichi Tani, Takanori Oyama, Hiroaki Sato, Kazuhiro Noma, Shunsuke Kagawa, Hisataka Kobayashi, Takuo Noda, Toshiyoshi Fujiwara. Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3831.

Purpose The cause of pectus excavatum has been hypothesized to be overgrowth of the costal cartilage. According to this theory, the length of costal cartilages must be longer in the side of deep depression in asymmetric patients. To challenge this hypothesis, we measured the lengths of ribs and costal cartilages and investigated lateral differences. Subjects and methods Twenty-four adolescent and adult patients with asymmetric pectus excavatum (14-30 years of age) with no history of surgery were investigated in this study. The fifth and sixth ribs and costal cartilages were individually traced to measure their full lengths on 3-dimensional computed tomographic (CT) images. As an index of asymmetry, sternal rotation angle was measured in the chest CT images. Patients with a 21° or greater angle of sternal twist were designated as an asymmetric group and those with an angle of smaller than 20° as a symmetric group. Lateral differences in the fifth and sixth costal and costal cartilage lengths were compared between the groups. Results On comparison of the costal and costal cartilage lengths in the asymmetric group, the right fifth ribs and costal cartilages were significantly shorter than the left ( P = .02 and .03, respectively), and right sixth ribs were also significantly shorter than the left ( P = .004), but right sixth costal cartilages were not ( P = .31). In the symmetric group, the lengths of the left and right fifth ribs and costal cartilages were showing no significant difference ( P = .20 and P = .80, respectively), and those of the sixth ribs and costal cartilage were also showing no significant difference ( P = .97 and P = .64, respectively). Discussion The ribs and costal cartilages on the right side with severer depression were significantly shorter or not different than those on the contralateral side. Based on these findings, the theory of costal cartilage overgrowth is contradictory. The etiology of asymmetric chest deformity should be reevaluated.

Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Amplification and overexpression of the MYCN proto-oncogene occur in approximately 20% of NB. Since MYCN activation is highly associated with poor prognosis, MYCN is an attractive target for the treatment of MYCN-amplified NB. MYCN is a transcriptional activator that induces the expression of many down-stream target genes including human telomerase reverse transcriptase (hTERT). We developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific virus replication, and OBP-702 further expresses tumor suppressor p53 protein. Although hTERT-driven oncolytic viruses are expected to be effective against MYCN-amplified NB with hTERT activation, the therapeutic potential of these viruses in MYCN-amplified NB remains unclear. In this study, we investigated whether OBP-301 and OBP-702 induce cell death and modulate MYCN expression in MYCN-amplified NB cells. Methods: We used 3 human NB cell lines with MYCN amplification, including IMR-32, CHP-134 and LA-N-5. The antitumor effect of OBP-301 and OBP-702 was evaluated using XTT assay. Virus-mediated cell death and modulation of MYCN and E2F1 expression were analyzed by Western blot analysis. The mRNA expression of hTERT and MYCN was analyzed by real-time RT-PCR analysis. To explore the role of E2F1 in the virus-mediated MYCN modulation, Ad-E2F1, a replication-defective adenovirus expressing E2F1 gene, was further used. Results: All NB cell lines showed high hTERT mRNA expression. OBP-301 and OBP-702 showed profound antitumor effect through autophagy-related cell death in all NB cell lines. Both viruses induced E1A and its target mediator E2F1 expression in these cell lines. Expression of MYCN mRNA and protein was downregulated by these viruses. Replication-deficient Ad-E2F1 infection also downregulated the expression of MYCN. Conclusions: These results suggest that hTERT-driven oncolytic adenoviruses are promising antitumor agent for the treatment of MYCN-amplified NB. These viruses induce profound autophagy through hTERT-dependent viral replication. E2F1 upregulation by viral infection is supposed to be one of the causes for MYCN downregulation and autophagy-related cell death. In vivo experiments are under way to investigate the antitumor effect of these viruses against xenograft NB tumors. Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Ablation of oncogenic MYCN expression by hTERT-driven oncolytic adenovirus induces cell death in MYCN-amplified neuroblastoma [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 5816. doi:10.1158/1538-7445.AM2017-5816

Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. More than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis at the time of diagnosis. Moreover, even after curative surgery, PDAC patients still have poor prognosis due to local recurrence and systemic metastasis. Although activation of oncogenic KRAS is implicated in the initiation, promotion and progression of pancreatic cancer, the development of cancer treatment targeting KRAS remains unsuccessful. Moreover, tumor suppressor p53 is also frequently inactivated in PDAC patients. We recently developed two types of tumor-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702, which is modified OBP-301 to express tumor suppressor p53 protein. In this study, we investigated whether oncolytic adenoviruses inhibit cell viability, migration, invasion and KRAS-driven signaling pathway in PDAC cells. Methods: We used 4 PDAC cells (MIA PaCa-2, Capan-1, Panc-1, BxPC-3). The cell viability was examined by XTT assay. Migration and invasion properties were assessed using transwell chamber assay. The modulation of KRAS-driven signaling pathway was investigated by Western blot analysis. Results: OBP-301 induced moderate antitumor effect in MIA Paca-2, Capan-1 and BxPC-3 cells and strong antitumor effect in Panc-1 cells in a dose-dependent manner, whereas OBP-702 induced profound antitumor effect in all human PDAC cells, suggesting the broad spectrum of OBP-702's efficacy. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-301 and OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells, and the inhibitory effect of OBP-702 was stronger than that of OBP-301. We also found that oncolytic adenoviruses inhibited the expression of KRAS and KRAS-downstream target MEK/ERK proteins in high-invasive type PDAC cells. Conclusions: Our results suggest that oncolytic adenoviruses, OBP-301 and OBP-702, inhibit the survival and invasive phenotype of pancreatic cancer by suppressing KRAS-driven signaling pathway and reactivating p53-driven signaling pathway. In vivo experiments are under way to investigate the anti-tumor and anti-invasive effects of oncolytic adenoviruses using BxPC-3 xenograft tumors. The clinical trial of intratumoral administration of oncolytic adenoviruses in patients with invasive PDAC should be also warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Takeshi Ieda, Kazuya Kuwada, Terutaka Tanimoto, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion. [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 3748.

For laparoscopic surgery in pediatric patients, the initial trocar insertion is usually made employing open laparotomy to avoid injury to the viscera. It is safe but somewhat time-consuming for establishing a pneumoperitoneum, and requires additional sutures for anchoring or preventing air leakage. We devised a new initial trocar insertion technique employing an umbilical center incision to shorten the surgical duration and improve the esthetic appearance.Four hundred and thirty-one pediatric patients were indicated for this umbilical center insertion method (UCM) at 2 pediatric surgical departments. Patients suspected of having umbilical lesions (except for umbilical hernia) or extensive bowel adhesions were excluded.There was no complication associated with UCM. In almost all patients, a pneumoperitoneum was established within 1 minute. The wound was completely within the umbilical ring, and very satisfactory esthetically.We consider UCM to be a safe and easy procedure, and acceptable method of initial trocar insertion for pediatric laparoscopic surgery.

Purpose The etiology of pectus excavatum (PE) has not been clarified. In 1944, Sweet (Sweet RH. Pectus excavatum. Ann Surg 1944;119:922-934) mentioned about the possibility of the overgrowth of costal cartilage being involved. However, no additional report that supports his hypothesis is available. In this study, we investigated whether the overgrowth of costal cartilage was an actual cause of PE through measurement of the costal cartilage length in PE patients and healthy controls. Materials and Methods We investigated the length of the fifth and sixth costal cartilages and ribs in PE patients from reconstructed images of 3-dimensional computed tomography. To examine the relative costal cartilage length, we calculated the C/R ratio, defined as the quotient of the costal cartilage length divided by the adjacent rib length, and compared it between PE patients and healthy controls. Results In PE patients, the C/R ratios were not larger than in healthy controls at any level. At the left sixth, the C/R ratio was significantly smaller in patients than in the healthy control group. Discussion The results revealed that, in PE patients, relative costal cartilage lengths were not longer than in healthy controls. We conclude that the overgrowth of costal cartilage is not the etiology of PE.

A full-term newborn male infant presented with dyspnea and cleft lip and palate. He was thought to have esophageal atresia with tracheoesophageal fistula. He underwent bronchoscopy before operation that showed a laryngotracheoesophageal cleft (LTEC) type III. The left main bronchus originated from the lower esophagus. His diagnosis was communicating bronchopulmonary foregut malformation (CBPFM) type IA associated with LTEC type III. Enhanced chest computed tomographic scan showed the left pulmonary artery originated from the descending aorta. Staged operations were indicated. At first, reconstruction of the left pulmonary artery was done at 3 months of age. Then at 6 months of age, operations for LTEC (tracheoplasty and esophagostomy) and CBPFM left bronchoplasty were performed. Reconstruction of esophagus was performed at age of 1 year. He is now 3 years old and doing well with a mild degree of bronchomalacia. This is the first report of total reconstruction of CBPFM type IA associated with LTEC.