Yuki Inagaki

A Nationwide Survey of Hepatitis E Virus Infection and Chronic Hepatitis E in Liver Transplant Recipients in Japan

Background Recently, chronic hepatitis E has been increasingly reported in organ transplant recipients in European countries. In Japan, the prevalence of hepatitis E virus (HEV) infection after transplantation remains unclear, so we conducted a nationwide cross-sectional study to clarify the prevalence of chronic HEV infection in Japanese liver transplant recipients. Methods A total of 1893 liver transplant recipients in 17 university hospitals in Japan were examined for the presence of immunoglobulin G (IgG), IgM and IgA classes of anti-HEV antibodies, and HEV RNA in serum. Findings The prevalence of anti-HEV IgG, IgM and IgA class antibodies was 2.9% (54/1893), 0.05% (1/1893) and 0% (0/1893), respectively. Of 1651 patients tested for HEV RNA, two patients (0.12%) were found to be positive and developed chronic infection after liver transplantation. In both cases, HEV RNA was also detected in one of the blood products transfused at the perioperative period. Analysis of the HEV genomes revealed that the HEV isolates obtained from the recipients and the transfused blood products were identical in both cases, indicating transfusion-transmitted HEV infection. Interpretation The prevalence of HEV antibodies in liver transplant recipients was 2.9%, which is low compared with the healthy population in Japan and with organ transplant recipients in European countries; however, the present study found, for the first time, two Japanese patients with chronic HEV infection that was acquired via blood transfusion during or after liver transplantation.

Clinical Features of Hepatitis E Virus Infection in Ibaraki, Japan: Autochthonous Hepatitis E and Acute-on-Chronic Liver Failure

Hepatitis E caused by hepatitis E virus (HEV) is a serious public health concern in developing countries where HEV is mainly transmitted through contaminated water. Recently, in industrialized countries, autochthonous hepatitis E, a porcine zoonosis, has been increasingly recognized. In Japan, the number of national notifications of acute hepatitis E has increased since the introduction of anti-HEV IgA antibody measurement, covered by the national health insurance program, in 2011. In the past three years, we examined five patients of acute hepatitis or acute-on-chronic liver failure caused by HEV infection who presented various clinical courses in the southern area of Ibaraki prefecture in Japan. Of these patients, 78-year-old and 63-year-old male patients presented acute hepatitis E and recovered by only bed rest. The latter patient had a history of consuming grilled or undercooked pork and shellfish prior to the onset of hepatitis E. Among the five patients examined, the infection route was detected only in this patient. Of note, a 65-year-old female patient presented severe hepatitis associated with painless thyroiditis. The patient was diagnosed with probable autoimmune hepatitis and was successfully treated with prednisolone (40 mg/day). Lastly, 58-year-old and 62-year-old male patients, both of whom had a history of diabetes mellitus and alcoholic liver disease, developed acute-on-chronic liver failure, and the latter patient with pre-existing liver cirrhosis died due to liver failure. Thus, patients with clinical HEV infection who display multiple underlying diseases can develop acute-on-chronic liver failure. In conclusion, HEV infection manifests the diverse clinical courses.

Cetuximab delivery and antitumor effects are enhanced by mild hyperthermia in a xenograft mouse model of pancreatic cancer

Even with current promising antitumor antibodies, their antitumor effects on stroma‐rich solid cancers have been insufficient. We used mild hyperthermia with the intent of improving drug delivery by breaking the stromal barrier. Here, we provide preclinical evidence of cetuximab + mild hyperthermia therapy. We used four in vivo pancreatic cancer xenograft mouse models with different stroma amounts (scarce, MIAPaCa‐2; moderate, BxPC‐3; and abundant, Capan‐1 and Ope‐xeno). Cetuximab (1 mg/kg) was given systemically, and the mouse leg tumors were concurrently heated using a water bath method for 30 min at three different temperatures, 25°C (control), 37°C (intra‐abdominal organ level), or 41°C (mild hyperthermia) (n = 4, each group). The evaluated variables were the antitumor effects, represented by tumor volume, and in vivo cetuximab accumulation, indirectly quantified by the immunohistochemical fluorescence intensity value/cell using antibodies against human IgG Fc. At 25°C, the antitumor effects were sufficient, with a cetuximab accumulation value (florescence intensity/cell) of 1632, in the MIAPaCa‐2 model, moderate (1063) in the BxPC‐3 model, and negative in the Capan‐1 and Ope‐xeno models (760, 461). By applying 37°C or 41°C heat, antitumor effects were enhanced shown in decreased tumor volumes. These enhanced effects were accompanied by boosted cetuximab accumulation, which increased by 2.8‐fold (2980, 3015) in the BxPC‐3 model, 2.5‐ or 4.8‐fold (1881, 3615) in the Capan‐1 model, and 3.2‐ or 4.2‐fold (1469, 1922) in the Ope‐xeno model, respectively. Cetuximab was effective in treating even stroma‐rich and k‐ras mutant pancreatic cancer mouse models when the drug delivery was improved by combination with mild hyperthermia.

Acute infection by hepatitis E virus with a slight immunoglobulin M antibody response

The anti-hepatitis E virus (HEV) immunoglobulin (Ig) M antibody response is generally regarded as a useful marker for diagnosing primary infection. However, in some cases, this antibody is not detected during the acute phase of infection. An 81-year-old man with stable membranous nephropathy who presented with asymptomatic acute liver dysfunction came to our hospital. HEV RNA of genotype 3 was detected in his serum, and he was diagnosed with acute hepatitis E. According to an enzyme-linked immunosorbent assay, high-level positivity for anti-HEV IgG and IgA antibodies was observed, but the assay was negative for IgM antibody throughout the clinical course of infection. The patient was not immunosuppressed. We further investigated the presence of IgM antibody using two other polyclonal antibodies against human IgM as secondary antibodies and another recombinant ORF2 protein of genotype 3 as an immobilized antigen. IgM was weakly detected in the serum during the acute phase only by the test with the antigen of genotype 3. Multi-genotype antigens can detect a slight IgM antibody response; however, anti-HEV IgA is more useful in diagnosing primary HEV infection, particularly in cases with a low IgM antibody response.

Treatment with ribavirin for chronic hepatitis E following living donor liver transplantation: A case report

Dear Editors, Our group recently published the first nationwide survey about the chronic hepatitis E virus (HEV) infection among Japanese liver transplant (LT) recipients. Importantly, two liver transplant recipients were diagnosed with sustained HEV infection after living donor liver transplantation (LDLT) via contaminated blood transfusion. Here, we describe a first case of established chronic hepatitis E after LDLT that was successfully and safely treated with a 20-week course of ribavirin. A 60-year-old Japanese lady underwent right liver LDLT at the age of 59 years with her 29-year-old son as the donor, for decompensated primary biliary cirrhosis. The immediate postoperative course was uneventful and she was discharged on postoperative day (POD) 45. Her immunosuppression regimen which consists of tacrolimus and methylprednisolone was based on the institutional protocol. At the time the survey was conducted, a maintenance dose of 4 mg/day of methylprednisolone was continued, and the trough level of tacrolimus was maintained at approximately 4–7 ng/dL. The clinical course of this patient since LDLT is otherwise shown in Figure 1. The details of the diagnosis of chronic hepatitis E is briefly described: (i) through the survey, anti-HEV immunoglobulin G was incidentally detected to be high-titer positive on POD 255 accompanied by hightiter HEV RNA (5.9 log copies/mL) and mildly elevated liver enzymes; (ii) retrospective and prospective investigation showed HEV antibodies and HEV RNA were undetectable in the serum of the donor; and (iii) HEV RNA was detected in 1 out of 12 units of the fresh frozen plasma (FFP) transfused to the patient during LDLT. The HEV isolates (genotype 3) detected from the recipient and the FFP were considered to be the same by complete nucleotide sequence identity, and the transfused FFP was determined to be the infectious source. The blood donor of the FFP was retrospectively found to have developed acute hepatitis E, but already to be fully resolved. Subsequently, the patient was diagnosed as developing chronic HEV infection for more than 6 months. Liver biopsy was avoided based on the lower platelet count (3.4×10/μL), which was presumably attributed to the persisting splenomegaly. Liver damage was then assessed by transient elastography which was compatible withmild liver graft damage (7.5 kPa). Thereafter, we started ribavirin for the treatment of chronic hepatitis E. Ribavirin was administrated p.o. starting from lower dose of

Abstract 171: Adipose-derived mesenchymal stem cell (ADSC) has the differentiation capacity toward cancer associated fibroblast (CAF) and reproduce the morphology of the clinical tumor stroma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Objectives] Crosstalk between cancer cells and Cancer-Associated Fibroblast (CAF) plays a crucial role that comprises 3D organization of solid cancers. It seems general understanding that the main origin of CAF is circulating bone marrow derived Mesenchymal Stem Cell (BM-MSC), though locally pre-existing resident MSC of adipose tissue (AD-MSC) should partly contribute to CAF formation. The role of CAF and MSC to tumorigenesis remains controversial,since some studies reported cancer stimulating effect by externally added MSC or its potential differentiating into CAF, while others showed its suppressive effect. The capability of MSCs innate tropism for cancer, which enables us to apply it for the cellular delivery of anti-cancer molecules, has attracted much attention. One of the key issues should be whether externally added MSC would accepted as a member of cancer 3D organization, and would contribute to the cancer morphology. We tried to reveal whether AD-MSC, which is much convenient source as engineered MSC than BM-MSC, stimulates or inhibit tumor growth, and how MSC contribute to tumor stromal morphogenesis. [Materials & Methods] Experiment: To evaluate the growth advantage by adding MSC, and how MSC contribute to tumor stromal morphogenesis, two differently-originated ADSC cell lines transfected with GFP was mixed with human pancreatic-cancer cell line (Capan-1) at a rate of 3×106 /5×106 , and inoculated into the back subcutaneous resion of BALB/cAJcl-nu/nu mice. The tumor volume was calculated at Day4, Day7 and Day10 to assess the growth advantage of tumor mixed with ADSC. The mice was sacrificed at Day 10 and the histology of the resected tumor was evaluated with fluorescence microscopy and immunostaining procedure with anti-GFP antibody. [Results] Capan-1 formed significantly larger subcutaneous tumor by adding with both two AD-MSC cell lines(352mm3,265mm3) compared to control (70mm3) at Day10. In the tumor mixed with one ADSC cell line, cancer cells formed ductal stractures and contained fiber rich stroma with GFP positive fibroblasts among cancer ducts, mimicked the similar distribution to that in clinical solid cancer specimen. However, in the graft with the other ADSC cell line, cancer cells presented cord-like structures or deregulated cell proliferation, not formed ductal stractures. Though GFP positive cells could be observed, they thinly and rondamly distributed in the stroma. [Discussion] Both two AD-MSC plays promotive role in tumor growth, and one of which strongly contribute to the tumor stromal morphogenesis. Our AD-MSC could be regarded as a case which obviously differentiated into CAF, and would be a good candidate source for forecomming CAF therapy. Further studies revealing CAFs specific signature would helpful for providing stable AD-MSC source. Citation Format: Yuki Inagaki, Tatsuya Oda, Tomohiro Kurokawa, Ryoichi Miyamoto, Yasuyuki Kida, Nobuhiro Ohkohchi. Adipose-derived mesenchymal stem cell (ADSC) has the differentiation capacity toward cancer associated fibroblast (CAF) and reproduce the morphology of the clinical tumor stroma. [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 171. doi:10.1158/1538-7445.AM2014-171

Clinical outcomes of sorafenib treatment failure for advanced hepatocellular carcinoma and candidates for regorafenib treatment in real-world practice: Outcomes of HCC patients treated with sorafenib

As second‐line therapy, regorafenib has been shown to provide a survival benefit for patients with hepatocellular carcinoma (HCC) who progress on sorafenib. In this retrospective study, we assessed the clinical outcomes of sorafenib treatment failure with regard to second‐line chemotherapy.

O011 : A nationwide survey of hepatitis E virus infection in liver transplant recipients in Japan

Results: Infrequent intrahepatic total HBVDNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T cell responses were similar between “anti-HBc alone” individuals and HBV resolvers (Figure 1). Circulating HBV-memory B cell responses were detected in all “anti-HBc alone” individuals, consistent with an HBsAgspecific memory pool. After one HBV vaccine dose, increased antiHBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P =0.0226) (Figure 2). Conclusions: “Anti-HBc alone” individuals showed HBV-specific T cell and memory B cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

Abstract 195: CD44v9 expression in clinical pancreatic cancer and the gemcitabine plus sulfasalazine therapy against chemoresistant pancreatic cancer murine model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective: Chemoresistance associating CD44-positive cancer stem cells may presents a crucial problem in treating pancreatic cancer. Among the various isoforms of CD44, valiant 9 (CD44v9) have revealed especially implicated in tumor growth, invasion, and metastasis. Interaction of CD44v9 with the cystine transporter subunit xCT provide the ability of cancer cells to defend themselves against reactive oxygen species, mediating chemoresistance. Sulfasalazine (SSZ), which is a specific inhibitor of xCT-mediated cystine transport,could be a new therapeutic weapon fight against cancer stem cells. However, the extent of this remains unclear, suggesting the need for a histological evaluation of CD44v9 expression in clinical pancreatic cancer and the anti-tumor effect on pancreatic cancer of SSZ. Therefore, In this study, we analyze the expression of CD44v9 in a pancreatic cancer surgical specimen and examine the anti-tumor effect of SSZ and altered expression of CD44v9 by administration of GEM (GEM) with tumorgrafts. Methods: Experiment 1: A total of 70 clinical primary ductal adenocarcinoma of the pancreas were applied for immunohistochemical evaluation of CD44v9 expression. Randomly picked up 10 fields for each cases were analyzed for intensity of membrane staining and the percentage of stained cancer cells per field, followed by summing the scores using our own scoring system. Experiment 2: Three lines of created tumorgrafts from clinical pancreatic cancer. We administered GEM to the tumorgrafts and also scored the clinical specimens’ immunohistochemical evaluation by CD44v9. In addition, using one line, we divided the mice into two groups, a GEM group and a GEM + SSZ group, and repeated as above to determine the effect on GEM of SSZ. Results: Experiment 1: There was no correlation for prognosis. It was found that CD44v9-positive cells are present in high numbers. Experiment 2: In 1 line, which increased CD44v9-positive cell after GEM treatment,the GEM group showed tumor regression of 85% and the GEM+SSZ group showed tumor regression of 90%. Discussion: Result in Experiment 1 is reasonable, therefore, to consider the significance of the treatment target CD44vv9 as likely to be very high. Further, there was one line of tumorgrafts used, but not all percentages of CD44v9-positive cells increased markedly by GEM administration. This suggests the possibility that CD44v9 may be regarded as a marker of GEM resistance. In addition, a small but insignificant tumor regression effect on GEM of SSZ was found. We present evidence that for pancreatic cancer as well as other carcinomas, CD44v9-positive cells may be regarded as one of the markers of chemo resistance. Furthermore, since SSZ shows the antitumor effects of plus for GEM, we suggest that SSZ treatment is a possibility for a new treatment option. Citation Format: Tomohiro Kurokawa, Tatsuya Oda, Yuki Inagaki, Ryoichi Miyamoto, Yoshimasa Akashi, Nobuhiro Ohkohchi. CD44v9 expression in clinical pancreatic cancer and the gemcitabine plus sulfasalazine therapy against chemoresistant pancreatic cancer murine model. [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 195. doi:10.1158/1538-7445.AM2014-195

Abstract 2663: Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objective: Histopathological characteristics of pancreatic carcinoma are the presence of abundant stroma that prevent mAb diffusion and consequently hampers mAb from direct attacking cancer cells. With a strong conviction that additional tool that improve mAb penetration and diffusion in dense fibroblastic solid cancer should indispensable for better therapeutic effect, we focused and employed the simple but strongest modalities, heat. Heat might increase tumor blood flow and tumor vessel permeability, leading enhanced accumulation of anticancer mAb. In this study, we aimed the increased accumulation of mAb in mouse pancreatic subcutaneous models and also boosted antitumor effects by applying moderate heat. Methods: Heat generation was completed by hot bathtub method for subcutaneous tumor in nude mice leg. Three human pancreatic cell of different stromal amount were employed (abundant: Capan-1, moderate: BxPC-3, and scant: MIAPaCa-2). At the timing of each tumors growth up to 70mm3, Cetuximab (1 mg/kg) was systematically administrated via caudal vein, subsequently applying heat to tumors for 30 min at three different temperatures of 25°C (control temperature), 37°C (intraabdominal organs), or 41°C (hyperthermia) (n = 5 each). The amount of accumulated Cetuximab were quantified by the fluorescent intensity of Alexa 488 against anti-human IgG at 24 hr, followed by figuring up the intensity of one cancer cell by dividing the total intensity value by the number of a cancer cell nuclei stained with DAPI. Enhanced antitumor effects, among irradiating various heat doses, were evaluated by the tumor volume over time. Results: In Capan-1, the fluorescent intensity per a cancer cell at 25°C, 37°C, 41°C were 675, 1130, 2332 respectively. Similarly, those in BxPC-3 were 673, 1347, 1573, and in MIAPaCa-2 were 1632, 1921, 1949, respectively. In Capan-1 and BxPC-3, the mean tumor growth on day 40 were significantly inhibited at 37°C group (934, 189 mm3), and 41°C group (491, 121 mm3) in compared with 25°C group (1385, 470 mm3) respectively. In MIAPaCa-2, however, that on day 50 was not inhibited at 37 degrees group (109 mm3), and 41 group (151 mm3) as compared with 25 group (123 mm3). Discussion: We demonstrated that hyperthermia actually contributed to measurable add-on antitumor effect for two among three pancreatic cancer models. Intending to provide more add-on effect, we are now evaluating the combined effects of hyperthermia plus applying a cyclic tumor-penetrating peptide (iRGD), which improved anticancer mAb penetration to cancer cells by binding to integrin αv/β3 or β5 and to neuropillin-1 on cancer cells. We suggested that this novel combined strategy would remarkably contribute to the enhanced accumulation of anticancer mAb in tumors and to the enhanced antitumor effects. Citation Format: Ryoichi Miyamoto, Tatsuya Oda, Shinji Hashimoto, Yoshimasa Akashi, Tomohiro Kurokawa, Yuki Inagaki, Nobuhiro Ohkohchi. Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model. [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 2663. doi:10.1158/1538-7445.AM2014-2663