Michihiro Ishida

Molecular diagnosis and therapy for occult peritoneal metastasis in gastric cancer patients

To apply an individualized oncological approach to gastric cancer patients, the accurate diagnosis of disease entities is required. Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer, and the tumor-node-metastasis classification includes cytological detection of intraperitoneal cancer cells as part of the staging process, denoting metastatic disease. The accuracy of cytological diagnosis leaves room for improvement; therefore, highly sensitive molecular diagnostics, such as an enzyme immunoassay, reverse transcription polymerase chain reaction, and virus-guided imaging, have been developed to detect minute cancer cells in the peritoneal cavity. Molecular targeting therapy has also been spun off from basic research in the past decade. Although conventional cytology is still the mainstay, novel approaches could serve as practical complementary diagnostics to cytology in near future.

Trastuzumab-Based Photoimmunotherapy Integrated with Viral HER2 Transduction Inhibits Peritoneally Disseminated HER2-Negative Cancer

Peritoneal dissemination is the most frequent metastasis in gastric cancer and is associated with poor prognosis. The lack of particular target antigens in gastric cancer other than HER2 has hampered the development of treatments for peritoneal dissemination of gastric cancer. We hypothesized that HER2-extracellular domain (HER2-ECD) gene transduction combined with trastuzumab-based photoimmunotherapy (PIT) might provide excellent and selective antitumor effects for peritoneal dissemination of gastric cancer. In vitro, adenovirus/HER2-ECD (Ad/HER2-ECD) efficiently transduced HER2-ECD into HER2-negative gastric cancer cells. Trastuzumab-IR700 (Tra-IR700)–mediated PIT induced selective cell death of HER2-ECD–transduced tumor cells. Ad/HER2-ECD also induced homogenous expression of HER2 in heterogeneous gastric cancer cells, resulting in uniform sensitivity of the cells to Tra-IR700–mediated PIT. Anti-HER2 PIT integrated with adenoviral HER2-ECD gene transfer was applied in mice bearing peritoneal dissemination of HER2-negative gastric cancer. Intraperitoneal administration of Ad/HER2-ECD and Tra-IR700 with PIT inhibited peritoneal metastasis and prolonged the survival of mice bearing MKN45. Furthermore, minimal side effects allowed the integrated therapy to be used repeatedly, providing better control of peritoneal dissemination. In conclusion, the novel therapy of molecular-targeted PIT integrated with gene transfer technology is a promising approach for the treatment of peritoneal dissemination in gastric cancer. Mol Cancer Ther; 15(3); 402–11. ©2016 AACR.

Integrated fluorescent cytology with nano-biologics in peritoneally disseminated gastric cancer

Gastric cancer patients positive for peritoneal cytology are at increased risk of tumor recurrence, but although a certain proportion of cytology‐positive patients relapse rapidly with aggressive progression, others survive longer with conventional chemotherapies. This heterogeneity makes it difficult to stratify patients for more intensive therapy and poses a substantial challenge for the implementation of precision medicine. We developed a new approach to identify biologically malignant subpopulations in cytology‐positive gastric cancer patients, using a green fluorescent protein (GFP)‐expressing attenuated adenovirus in which the telomerase promoter regulates viral replication (TelomeScan, OBP‐401). The fluorescence emitted from TelomeScan‐positive cells was successfully quantified using a multi‐mode microplate reader. We then analyzed clinical peritoneal washes obtained from 68 gastric cancer patients and found that patients positive for TelomeScan had a significantly worse prognosis. In 21 cytology‐positive patients, the median survival time of those who were TelomeScan positive (235 days) was significantly shorter than that for those who were TelomeScan negative (671 days; P = 0.0062). This fluorescent virus‐guided cytology detects biologically malignant cancer cells from the peritoneal washes of gastric cancer patients and may thus be useful for both therapy stratification and precision medicine approaches based on genetic profiling of disseminated cells.

Training system for laparoscopy-assisted distal gastrectomy

PurposeLaparoscopy-assisted distal gastrectomy (LADG) is likely to become a standard procedure for gastric cancer, which highlights the importance of establishing a training system in which even inexperienced surgeons can perform this procedure safely. This study assesses our training system for LADG based on short-term surgical outcomes.MethodsWe evaluated retrospectively the short-term outcomes of 100 consecutive LADGs with curative D1/D1+ lymph node dissection. Our training system was assessed based on the learning curve of trainees, and factors related to achieving good-quality operations were analyzed statistically.ResultsOverall, postoperative complications developed in 10 patients (10%), and included one case of anastomotic leakage (1%) and one case of pancreatic fistula (1%). The learning curve of the trainees plateaued after 10 operator cases in terms of operation time. The importance of the trainer’s position was also confirmed by the result that the operation time was significantly longer when trainees with ≤10 operator cases performed LADG with a trainer as scopist vs. a trainer as the first assistant. Univariate and multivariate analyses revealed that >10 operator cases were the most important factor for achieving good-quality operations.ConclusionThese results show that our current LADG procedure and training system are appropriate and effective.

Abstract 3412: Virus-guided fluorescence imaging of intraperitoneal free gastric cancer cells: a preliminary clinical study as a potential clinical biomarker

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objectives: In patients with gastric cancer, peritoneal dissemination is the most common metastasis. To predict future peritoneal recurrences, peritoneal lavage cytology is performed during operation. But even cytology-negative patients sometimes develop peritoneal recurrences. Thus an additional method to detect intraperitoneal free gastric cancer cells is necessary. We have developed a genetically engineered adenovirus, TelomeScan, which replicates and expresses GFP only in telomerase-activated cancer cells. Here we detected intraperitoneal free gastric cancer cells using TelomeScan, and investigated the correlation between the number of GFP-positive cells and patient prognosis. Methods: Peritoneal wash was obtained from 69 gastric cancer patients during operation. The cells in the wash were infected with TelomeScan for 24 hours. Finally, GFP-positive cells were counted under a fluorescence microscope. In some GFP-positive cases immunofluorescence assay was added. Clinicopathological data were obtained from medical records. Then we examined different cut-off values (the number of GFP-positive cells which indicates TelomeScan-positive) and estimated survival curves using the Kaplan-Meier method, and compared using the Wilcoxon test. Results: For a cut-off value of 10, 25 of the 69 cases were TelomeScan-positive (10 or more GFP-positive cells). And these 25 cases showed the most significant worse prognosis when compared to the 44 TelomeScan-negative cases (p = 0.0040). In addition, 17 of the 69 cases were conventional cytology-positive. Of these 17 cases, 9 were TelomeScan-positive, and these 9 cases showed significantly worse prognosis than the 8 TelomeScan-negative conventional cytology-positive cases (p = 0.0017, MST 195 days). Under fluorescence microscope we observed that GFP-positive cells sometimes formed cell clusters with GFP-negative cells. Immunofluorescence assay showed that these GFP-negative cells expressed CD45, which means these cells were leukocytes. Conclusion: We have successfully detected cancer cells in peritoneal wash using TelomeScan. The presence of GFP-positive cells in peritoneal wash was associated with worse prognosis. TelomeScan-positive patients, especially in conventional cytology-positive cases, showed remarkably worse prognosis than TelomeScan-negative conventional cytology-positive patients. Our data suggest that TelomeScan-guided cytological detection may have clinical implications as a prognostic biomarker in gastric cancer. Citation Format: Megumi Watanabe, Shunsuke Kagawa, Kazuya Kuwata, Michihiro Ishida, Yuuri Hashimoto, Naoto Hori, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Hiroshi Tazawa, Yasuo Urata, Toshiyoshi Fujiwara. Virus-guided fluorescence imaging of intraperitoneal free gastric cancer cells: a preliminary clinical study as a potential clinical biomarker. [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 3412. doi:10.1158/1538-7445.AM2015-3412

Abstract 4726: Virus-guided fluorescence imaging of intraperitoneal free gastric cancer cells as a potential clinical biomarker

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Peritoneal dissemination is a common pattern of metastasis in patients with gastric cancer, and associated with worse prognosis. Peritoneal lavage cytology during the operation is an important factor in predicting future development of peritoneal metastasis and determining the treatment strategy, but its sensitivity and specificity are unsatisfactory. TelomeScan is an adenovirus engineered to replicate and express GFP only in telomerase-activating tumor cells, so that we can easily detect viable cancer cells even among numerous normal cells. We hypothesized that TelomeScan might be applicable to detecting free cancer cells in peritoneal wash. Methods: Peritoneal washes were obtained from 42 gastric cancer patients during operation. The number of GFP-positive cells was determined for each sample and compared with cytology results and clinicopathological data Results: Clinical stage was ranged from IA to IV, and thirteen cases were diagnosed as class IV or class V by peritoneal lavage cytology. More than 10 GFP-positive cells were detected in 12 out of 42 cases, and these cases showed a worse prognosis when compared to the other 30 cases. Conclusion: We were able to detect gastric cancer cells as GFP-positive cells in peritoneal wash using TelomeScan. Furthermore, the presence of GFP-positive cells in peritoneal wash was associated with worse prognosis. These results suggest that number of cancer cells detected by TelomeScan in the peritoneal wash may have important clinical implication as prognostic and therapeutic biomarkers in gastric cancer. Citation Format: Megumi Watanabe, Shunsuke Kagawa, Michihiro Ishida, Naoto Hori, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Hiroshi Tazawa, Toshiyoshi Fujiwara. Virus-guided fluorescence imaging of intraperitoneal free gastric cancer cells as a potential clinical biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4726. doi:10.1158/1538-7445.AM2014-4726

Abstract 2615: Novel combination therapy of adenoviral gene transfer of HER2-extracellular domain and trastuzumab-based photoimmunotherapy for HER2 negative cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Humanized anti-HER2 monoclonal antibody, trastuzumab, has provided survival benefit to patients with HER2 positive breast cancer and advanced gastric cancer. However, the treatment with trastuzumab was limited due to low or heterogeneous HER2 positivity, insufficient effects as single agent and acquisition of resistance. To overcome these problems, we employed the adenoviral vector expressing HER2-extracellular domain (HER2-ECD), Ad/ HER2-ECD, which is designed to tag cancer cells with HER2-ECD and to sensitize them to trastuzumab, and a novel photoimmunotherapy (PIT). PIT consists of trastuzumab-conjugated with a photosensitizer, IR700 (Tmab-IR700) and irradiation with near-infrared light, induces a strong cytotoxicity on HER2 positive cancer cells. We assessed the effect of Ad/HER2-ECD and Tmab-IR700 mediated PIT in HER2 negative cancer cells. Methods: HER2 negative breast cancer cells (MCF7, MDA-MB-231) and gastric cancer cells (MKN1 and MKN45) were infected with Ad/ HER2-ECD. They were treated with Tmab-IR700 mediated PIT. We evaluated the HER2-ECD expression induced by Ad/ HER2-ECD and the cytotoxic effects of Tmab-IR700 mediated PIT. Results: Ad/HER2-ECD efficiently expressed HER2 protein on the surface of the cancer cells. HER2-ECD expressing cells were selectively and rapidly killed by Tmab-IR700 mediated PIT, which was morphologically captured as bleb formation of cellular membrane. Conclusions: We demonstrated that Ad/ HER-ECD combined with Tmab-IR700 mediated PIT could induce target-selective, immediate cell death in HER2 negative cancer cells. The combination therapy of adenoviral gene transfer of HER2-ECD and the molecular-target PIT is effective for cancer cells regardless of HER2 expression. Citation Format: Michihiro Ishida, Shunsuke Kagawa, Kyoko Shimoyama, Kiyoto Takehara, Kazuhiro Noma, Shunsuke Tanabe, Hiroshi Tazawa, Junji Matsuoka, Hisataka Kobayashi, Toshiyoshi Fujiwara. Novel combination therapy of adenoviral gene transfer of HER2-extracellular domain and trastuzumab-based photoimmunotherapy for HER2 negative cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2615. doi:10.1158/1538-7445.AM2014-2615