Motohiko Tokuhisa

Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer

Peritoneal metastasis is the most frequent type of recurrence in patients with gastric cancer (GC) and is associated with poor prognosis. Peritoneal lavage cytology, used to evaluate the risk of peritoneal metastasis, has low sensitivity. Here, we assessed the diagnostic potential of exosomal miRNA profiles in peritoneal fluid for the prediction of peritoneal dissemination in GC. Total RNA was extracted from exosomes isolated from six gastric malignant ascites (MA) samples, 24 peritoneal lavage fluid (PLF) samples, and culture supernatants (CM) of two human gastric carcinoma cell lines that differ in their potential for peritoneal metastasis. Expression of exosomal miRNAs was evaluated with Agilent Human miRNA microarrays and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microarray analysis indicated a low variability in the number and signal intensity of miRNAs detected among the samples. In the six MA fluids, miR-21 showed the highest signal intensity. We identified five miRNAs (miR-1225-5p, miR-320c, miR-1202, miR-1207-5p, and miR-4270) with high expression in MA samples, the PLF of serosa-invasive GC, and the CM of a highly metastatic GC cell line; these candidate miRNA species appear to be related to peritoneal dissemination. Differential expression of miR-21, miR-320c, and miR-1225-5p was validated in the PLF of serosa-invasive and non-invasive GC by qRT-PCR and miR-21 and miR-1225-5p were confirmed to be associated with serosal invasion in GC. PLF can be used to profile the expression of exosomal miRNAs. Our findings suggest that miR-21 and miR-1225-5p may serve as biomarkers of peritoneal recurrence after curative GC resection, thus providing a novel approach to early diagnosis of peritoneal dissemination of GC.

Effect of obesity on laparoscopy-assisted distal gastrectomy compared with open distal gastrectomy for gastric cancer

This study compared surgical outcomes between patients undergoing laparoscopy‐assisted distal gastrectomy (LADG) and those undergoing open distal gastrectomy (ODG) from the viewpoint of obesity.

Sorafenib Monotherapy in a Patient with Unresectable Hepatic Epithelioid Hemangioendothelioma

A 49-year-old Japanese man had multiple huge masses (max. size 60 mm diameter) in his liver. These tumors were pathologically diagnosed by tumor biopsy as epithelioid hemangioendotheliomas of the liver. In this case, multiple liver tumors existed in both lobes. Also this patient did not agree to receive surgical resection including liver transplantation. Chemotherapy with sorafenib at a dose of 400 mg/body twice a day was started. About 6 months later, CT findings revealed that these tumors were shrinking slightly; 33 months later, the tumors obviously showed a partial response in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST). Also 60 months later, the partial response continued with sorafenib monotherapy.

Effect of Folfirinox as second-line chemotherapy for metastatic pancreatic cancer after gemcitabine-based chemotherapy failure

Background: This study aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicity, and efficacy of second-line chemotherapy with FOLFIRINOX after gemcitabine (GEM)-based chemotherapy failure in metastatic pancreatic cancer (MPC). Methods: We studied 18 histopathologically proven MPC patients. The schedule was 85 mg/m2 oxaliplatin, irinotecan, and 400 mg/m2 leucovorin, followed by 400 mg/m2 5-fluorouracil (5-FU) as a bolus on day 1 and 2400 mg/m2 5-FU as a 46-hour continuous infusion biweekly. The dose of irinotecan was defined as follows: level 0: 100 mg/m2, level 1: 125 mg/m2, level 2: 150 mg/m2, and level 3: 180 mg/m2. The doses of other drugs were fixed. The primary endpoint of phase II study was the response rate (RR). Results: We initially evaluated 6 patients in a phase I study. One patient developed neutropenia and 1 patient developed hyperglycemia and severe infection. Accordingly, level 1 was chosen as the MTD. According to a phase II study, the RR was 22.2% and the disease control rate was 61.1%. The progression-free survival and overall survival were 2.8 (range, 0.7–19.1) and 9.8 (2.4–19.8) months, respectively. The most common severe adverse event was neutropenia (66.7%). Febrile neutropenia occurred in 1 (5.6%) case. Conclusion: The recommended dose was 85 mg/m2 oxaliplatin, 100 mg/m2 irinotecan, and 400 mg/m2 leucovorin, followed by 400 mg/m2 5-FU as a bolus on day 1 and 2400 mg/m2 5-FU as a 46-hour continuous infusion. These results indicate that second-line FOLFIRINOX is a marginally effective treatment for GEM-based chemotherapy failure cases.

Low-dose docetaxel and cisplatin combination chemotherapy for stage II/III gastric cancer showing resistance to S-1 adjuvant chemotherapy: a phase I study

Abstract To establish a safe, long-term regimen of docetaxel (DOC) and cisplatin (CDDP) in an outpatient setting for gastric cancer refractory to S-1 adjuvant chemotherapy, a dose-escalating phase I study was conducted. Cohorts of patients were treated with escalating doses of DOC (starting at 20 mg/m2 per week with 5 mg/m2 increments) and a fixed dose of CDDP (25 mg/m2). Drugs were administered on days 1, 8, and 15. A cycle of this treatment was 28 days. In total, 52 courses were performed, and the mean number of courses was 5·3. Two of the four patients at dose level 3 showed dose-limiting toxicities (grade 4 neutropenia, and grade 3 anorexia and dehydration). The recommended dose (RD) of DOC was therefore defined as 25 mg/m2. There is a need for a phase II clinical trial using this regimen in patients with S-1-refractory stage II/III gastric cancer.

Clinical Usefulness of Somatostatin Receptor Scintigraphy in Japanese Patients with Gastroenteropancreatic Neuroendocrine Tumors.

Background/Aims: Somatostatin receptor (SSTR) scintigraphy (SRS) is the standard imaging modality for evaluation of gastroenteropancreatic neuroendocrine tumor (GEP-NET) in Western countries. However, this modality was not approved in Japan until recently. The purpose of this study was to evaluate the clinical efficacy of SRS for detecting GEP-NET in Japanese patients. Methods: Japanese patients with advanced GEP-NET were enrolled and evaluated by the SRS and CT. We also compared SRS and immunohistochemical expression of SSTR type 2a (SSTR2a). Results: We enrolled 16 patients and the primary sites were the pancreas in 9, the stomach in 1, the small intestine in 2, the colon in 3, and unknown in 1. SRS showed positive findings in 3 (100%) of grade 1 (G1) and in 12 (92.3%) of grade 2 (G2) lesions. In the liver, SRS and CT detected lesions in 13 and 14 cases, respectively. The concordance rate of SSTR2a expression with SRS findings was 93.8% in the whole body and 92.9% in the liver. Conclusions: SRS could detect almost all of G1 and G2. SRS could be useful to detect lesions, with a high concordance rate with CT and pathological findings. We confirmed that SRS is a useful and reliable modality for Japanese patients.

Abstract 3134: Pilot study of immune status of GEP-NETs in tumor microenvironment

Introduction: Neuroendocrine tumor (NET) is a rare cancer, however, morbidity rate is increasing every year. Recently, effective molecular targeting drugs for NET were developed and leading acceptable results, however, it is still not enough. Immune checkpoint inhibitor is becoming a very effective drug in malignant melanoma, lung cancer, and some kind of cancer showing microsatellite instability (MSI). Aim: Aim of this study is to investigate NET about MSI status and immune status of microenvironment using NET patients9 operative or biopsy samples. Method: Patients were 20 NETs, male: 11, female: 9, G1: 6, G2: 8, G3: 6, pancreas: 11, duodenum: 4, colon: 2, liver: 1, unknown: 2. Using patients9 samples by operative resection or biopsy, examinations by immunohistochemistry were performed to confirm MSI status using MSH2, MSH6, PMS2 and MLH1 antibody and count the cell number expressing PD-L1, PD-1, CD8 or Foxp3. Expression of MSH2, MSH6, PMS2, MLH1 and PD-L1 was evaluated on tumor cells. Specimens were categorized as IHC negative or positive if 1% of cells were stained by each monoclonal antibody. Expression of PD-1, CD8, Foxp3 was evaluated on the infiltrating lymphocyte of intratumor and peritumor respectively. Positive cells absolute number of each staining were counted in the 3 hot spot field (X400) and then the average of the 3 field of each specimen was utilized. Results: MSI: Expression of the 4 mismatch repair protein could be detected in the all 20 NETs tumor, then there was no case showing MSI. PD-L1 was detected in the 15 cases (75%). PD-L1 was expressed in the surface of tumors. PD-1 was also detected 15 cases (75%). PD-1 was expressed in the surface of infiltrative intratumoral and peritumoral lymphocyte. Both of PD-L1 in the tumor and PD-1 in the lymphocyte were detected in the 12 cases (60%). PD-L1 was detected in 72.7% of pancreatic NET, 72% of gastroenteral NET. PD-L1 was detected in 100% of NET-G1, 75% of G2 and 50% of G3. The absolute number of intratumoral PD-1+ lymphocyte, CD8+ lymphocyte or Foxp3+ lymphocyte was higher in the PD-L1+ tumor than in the PD-L1- tumor. In peritumor, there was not a similar tendency. In the PD-L1 positive NET, the absolute number of intratumoral PD-1+ lymphocyte or CD8+ lymphocyte increased according to NET grade. Conclusion: Seventy-five% of GEP-NET expressed PD-L1. It might be higher rate of PD-L1 expression compared with other cancers. The absolute number of Intratumoral infiltrating lymphocyte expressing PD-1 or CD8 was also high. There might be a fraction of a good target for immune check point therapy in NETs. Citation Format: Yasushi Ichikawa, Noritoshi Kobayashi, Ayumu Goto, Motohiko Tokuhisa, Yukihiko Hiroshima, Takashi Ishikawa, Shoko Takano, Tomio Inoue, Itaru Endo. Pilot study of immune status of GEP-NETs in tumor microenvironment [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 3134.

Abstract CT318: FOLFOXIRI+B-mab showed powerful effect as preoperative chemotherapy for multiple liver metastases of colorectal cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background; R0 resection for liver metastases of colorectal cancer is one of the promising treatment to improve prognosis of advanced colorectal cancer. Powerful regimen FOLFOXIRI including 3 cytotoxic drugs; 5FU, irinotecan and oxaliplatin was reported as promising treatment for advanced colorectal cancer (J Clin Oncol 2007; 25: 1670-76). In our department Phase I/II study of FOLFOXIRI+B-mab including fluoroiuracil/oxaliplatin/irinotecan/bevacizumab for advanced liver metastases of colorectal cancer as pre-operative chemotherapy has been conducted now, and Phase II was on going. In this study, we report effect and safety of FOLFOXIRI+B-mab. Methods: The study was designed as a single-arm, open-label phase I/II trial. Phase I was conducted to decide tolerated dose (MTD) of irinotecan and 150 mg/m2 was decided as recommended dose. Patients who are colorectal cancer with 4 or more liver metastases and no other distant metastases are included. The regimen includes bevacizumab; 5 mg/kg, irinotecan; 150 mg/m2, oxaliplatin; 85 mg/m2, l-LV; 200 mg/m2, 5FU; 400 mg/m2 administered on day 1, followed by 5FU; 2400 mg/m2 continuously administered for 46 hours. R0 resection rate of liver metastases as primary endpoint will be evaluated. Results: Currently, 16 patients were enrolled. Two patients dropped out because of grade 3 diarrhea and grade 3 thromboembolism. One patient is waiting for liver resection and R0 resection of liver metastases was performed for 9 patients. In the 14 patients excluding 2 drop out, 13 showed PR and average of reduction rate was 51%. There was no pathological CR in the 9 patients who was performed R0 resection. There was no severe postoperative complication in them. Conclusion: This regimen is safe and shows high PR rate. So, the regimen is effective and improve R0 resection rate for multiple liver metastases of colorectal cancer. Further investigation of this study is still ongoing now. Citation Format: Yasushi Ichikawa, Ayumu Goto, Noritoshi Kobayashi, Motohiko Tokuhisa, Takashi Ishikawa, Atsushi Ishibe, Kazuteru Watanabe, Kazunori Nojiri, Yoshibumi Kumamoto, kazuhisa Takeda, Mitsuyoshi Ota, Hirotoshi Akiyama, Kuniya Tanaka, Itaru Endo. FOLFOXIRI+B-mab showed powerful effect as preoperative chemotherapy for multiple liver metastases of colorectal cancer. [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 CT318. doi:10.1158/1538-7445.AM2014-CT318

Abstract 5279: Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. A microRNA (miRNA) is non-coding RNA molecules of 21-25nt. miRNAs are post-transcriptional regulators that bind to messenger RNA transcripts, resulting in translational repression of target degradation and gene silencing. miRNAs are wrapped in exosome and secreted stably in body fluid, which can prevent RNase from degrading the miRNAs. Therefore several methods for miRNA-based early cancer detection using serum, plasma, and urine are reported. However there is no report to detect peritoneal dissemination of gastric cancer using malignant ascitis. Aim. To detect predictive factors for peritoneal dissemination of gastric cancer, global analysis of exosomal miRNAs in gastric malignant ascites, intraoperative peritoneal lavage fluid and cell culture supernatant were performed. Materials and Methods. A total of 6 gastric malignant ascites, 20 peritoneal lavage fluids and 2 human gastric carcinoma cell culture supernatants (OCUM-2M and its sub line with high potential for metastasis to the peritoneal cavity named OCUM-2MD3) were included in this study. The amount of 10 ml samples underwent a final ultracentrifugation at 120,000g twice to pellet the exosomes. miRNA was extracted and analyzed using Bioanalyzer. Exosomal miRNA expression profiling was performed using the Agilent Human miRNA Microarrays. To quantitatively detect mature miRNAs, TaqMan MicroRNA Assays was used. Statistical tests by spearmans rank-correlation coefficient were performed to investigate correlation of exosomal miRNAs that were expressed between malignant ascites and cell culture supernatant. Results. miRNAs were detected in each gastric malignant ascites at concentrations ranging from 61.7 ng to 220 ng, however no or very low amounts of ribosomal RNA (18S and 28S). Microarray analysis detected 327 miRNAs in malignant ascites, 389 in OCUM-2M and 414 in OCUM-2MD3. miRNA expression of Malignant ascites correlated that of cell culture supernatants; OCUM-2M(r=0.774, p 2-fold change) than in OCUM-2M. Among these 140 miRNAs, 7 miRNAs detected at high expression in malignant ascites were selected for confirmation by qRT-PCR. All 7 miRNA was detectable both malignant ascites and intraoperative peritoneal lavage fluids by qRT-PCR. Conclusions. Exosomal miRNAs were highly detectable in malignant ascites and peritoneal lavage fluids. In this study 7 miRNAs related to peritoneal dissemination were identified. This result may provide a new diagnostic approach of peritoneal dissemination. Citation Format: Motohiko Tokuhisa, Yasushi Ichikawa, Takashi Kosaka, Satoshi Hasegawa, Hirotoshi Akiyama, Takashi Ishikawa, Nobuyoshi Kosaka, Takahiro Ochiya, Masakazu Yashiro, Kousei Hirakawa, Chikara Kunisaki, Ayumu Goto, Noritoshi Kobayashi, Itaru Endo. Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2013-5279 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.