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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.

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.

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.