Kotarosumitomo Nakayama

Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem around the world. NAFLD patients with nonalcoholic steatohepatitis (NASH) can develop cirrhosis and hepatocellular carcinoma. The ability to distinguish NASH from simple steatosis would be of great clinical significance. Ballooning hepatocytes are characteristic of typical pathological NASH; here, the polarized secretion of proteins is disrupted due to destruction of the cytoskeleton. We previously reported that fucosylated glycoproteins are secreted into bile, but not into sera in normal liver. Therefore, we hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes, and serum fucosylated glycoproteins would increase in NASH patients. To confirm our hypothesis, we evaluated serum fucosylated haptoglobin (Fuc-Hpt) levels in biopsy-proven NAFLD patients (n = 126) using a lectin-antibody ELISA kit. Fuc-Hpt levels were significantly increased in NASH patients compared with non-NASH (NAFLD patients without NASH) patients. Interestingly, Fuc-Hpt levels showed a significant stepwise increase with increasing hepatocyte ballooning scores. Multiple logistic regression analysis showed that Fuc-Hpt levels were independent and significant determinants of the presence of ballooning hepatocytes. Moreover, Fuc-Hpt levels were useful in monitoring liver fibrosis staging. Next, to investigate the significance of serum Fuc-Hpt in a larger population, we measured Fuc-Hpt levels in ultrasound-diagnosed NAFLD subjects (n = 870) who received a medical health checkup. To evaluate NAFLD disease severity, we used the FIB-4 index (based on age, serum AST and ALT levels, and platelet counts). Fuc-Hpt levels increased stepwise with increasing FIB-4 index. Conclusion Measurement of serum Fuc-Hpt levels can distinguish NASH from non-NASH patients, and predict the presence of ballooning hepatocytes in NAFLD patients with sufficient accuracy. These results support the potential usefulness of measuring Fuc-Hpt levels in clinical practice.

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes

AIM To evaluate/isolate cancer stem cells (CSCs) from tissue or cell lines according to various definitions and cell surface markers. METHODS Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. RESULTS Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabine-resistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. CONCLUSION Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.

Mutation of GDP-Mannose-4,6-Dehydratase in Colorectal Cancer Metastasis

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.

Establishment of a novel lectin–antibody ELISA system to determine core-fucosylated haptoglobin

BACKGROUND Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker that increases in various pathological conditions. We previously established a Fuc-Hpt lectin-antibody assay using Aleuria aurantia lectin (AAL), and applied this to diagnose several diseases, including various cancers. AAL recognizes both α1-3/1-4 and α1-6 fucosylation on N/O-linked glycans. These fucosylation types differ in biological function, and in regulation by different fucosyltransferases. Recently, we identified a novel lectin, Pholiota squarrosa lectin (PhoSL), which specifically recognizes α1-6 fucosylation (core-fucosylation). METHODS We developed a lectin-antibody ELISA kit using PhoSL to determine core-Fuc-Hpt levels in sera from colorectal or pancreatic cancer patients. RESULTS Serum levels of AAL-reactive Hpt are higher in pancreatic cancer patients, whereas those of PhoSL-reactive Hpt are higher in colorectal cancer patients. Mass spectrometry analyses of Hpt fucosylation levels were consistent with lectin-antibody ELISA results. Hpt-transfected colorectal cancer cell lines produced significant amounts of core-Fuc-Hpt, suggesting that colorectal cancer tissues produce core-Fuc-Hpt. CONCLUSIONS These differences in Fuc-Hpt patterns might depend on cancer cells and the surrounding cells, which produce Hpt.

Serum Mac-2 binding protein is a novel biomarker for chronic pancreatitis

AIM To determine the efficacy of Mac-2 binding protein (Mac-2bp) for diagnosis of chronic pancreatitis. METHODS Fifty-nine healthy volunteers (HV), 162 patients with chronic pancreatitis (CP), and 94 patients with pancreatic ductal adenocarcinoma (PDAC) were enrolled in this study. We measured serum Mac-2bp using our developed enzyme-linked immunosorbent assay kit. Additional biochemical variables were measured using an automated analyzer (including aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, triglyceride, C-reactive protein, and amylase levels) or chemiluminescent enzyme immunoassay (carbohydrate antigen 19-9 and carcinoembryonic antigen). The ability of Mac-2bp to predict CP diagnosis accurately was assessed using receiver operating characteristic (ROC) analyses. RESULTS Serum Mac-2bp levels were significantly increased in CP patients compared to HV (P < 0.0001) and PDAC patients (P < 0.0001). Area under the ROC curve values of Mac-2bp for the discrimination of CP from HV and PDAC were 0.727 and 0.784, respectively. Multivariate analyses demonstrated that serum Mac-2bp levels were independent determinants for CP diagnosis from HV and PDAC patients. Immunohistological staining showed that Mac-2bp was expressed faintly in the pancreas tissues of both CP and PDAC patients. Serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, and triglyceride levels were significantly higher in patients with CP or PDAC. Serum Mac-2bp levels were highly correlated with protein levels of alanine aminotransferase, γ-glutamyltransferase, and C-reactive protein, but not amylase, suggesting that the damaged liver produces Mac-2bp. CONCLUSION Measurement of serum Mac-2bp may be a novel and useful biomarker for CP diagnosis as well as liver fibrosis in the general population.

Distinct effects of daratumumab on indirect and direct antiglobulin tests: a new method employing 0.01 mol/L dithiothreitol for negating the daratumumab interference with preserving K antigenicity (Osaka method): 0.01 MOL/L DTT FOR THE DARA INTERFERENCE

There is an increasing demand for daratumumab (DARA), an immunoglobulin (Ig)G1κ monoclonal antibody (MoAb) that recognizes CD38, to manage relapsed or refractory multiple myeloma (MM) patients. However, DARA leads to positive and panreactive agglutination reactions in indirect antiglobulin tests (IATs) in vitro (the DARA interference). In addition, effects of DARA on red blood cells (RBCs) in vivo remains elusive.

Haptoglobin phenotype is a critical factor in the use of fucosylated haptoglobin for pancreatic cancer diagnosis

Fucosylation is one of the most important glycosylations involved in cancer and inflammation. Many studies have reported significant increases in serum fucosylated haptoglobin (Fuc-Hpt) in a variety of cancer patients. In this study, we measured Fuc-Hpt using a lectin-antibody enzyme-linked immunosorbent assay (ELISA) or a novel ELISA system that used a glycan antibody for Fuc-Hpt. Hpt is known to be divided into three phenotypes (Hpt1-1, Hpt2-1, and Hpt2-2), depending on its genetic background. Normal levels of serum Hpt are different in each Hpt phenotype and these phenotypes are associated with the incidence of several human diseases. Here, we investigated how Hpt phenotype affected measurements of Fuc-Hpt, using two kinds of ELISA. Interestingly, we found that serum Fuc-Hpt levels were dramatically lower in the Hpt1-1 phenotype for both types of ELISA. For Hpt2-1 and Hpt2-2, we observed significantly increased serum Fuc-Hpt levels in patients with pancreatic cancer. When cases of the Hpt1-1 phenotype were depleted, our receiver operating characteristic (ROC) curve analyses showed that the area under the curve (AUC) value for pancreatic cancer diagnosis increased in each ELISA. Taken together, our results indicate that Hpt phenotype is a critical for the clinical application of Fuc-Hpt as a cancer biomarker.

Abstract 3122: A strategy for detecting high-risk groups for pancreatic cancer with glycol-biomarkers

(Aim) Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers, showing a high potential for invasive activity and high recurrence rates. One of the reasons for poor prognosis of PDAC is a difficulty in an early diagnosis. When pancreatic tumors are detected by CT or MRI examinations, most cases are at the advanced stage even if tumor size is within 2 cm. To make an early diagnosis for PDAC truely, we should make a targeting of high-risk groups for developing PDAC like chronic hepatitis followed by hepatocellular carcinoma. Aberrant glycosylation is a promising target for cancer biomarkers. Several kinds of glyco-biomarkers are clinically used for PDAC diagnosis like CA19-9. We have reported that fucosylated haptoglobin (Fuc-Hpt) is a novel type of glyco-biomarker for PDAC. Serum Fuc-Hpt levels are dramatically increased at the stage IV of PDAC as well as a few cases of Stage I-III. In the present study, we performed pathological analysis of surrounding tissues around pancreatic cancer as well as normal pancreas in autopsy cases and investigated the clinical usefulness of glycol-biomarkers for chronic pancreatitis and pancreatic cancer (Subjects and Methods) 88 cases of pancreatic cancer patients and 100 cases of other diseases are enrolled in this study. Inflammation, fibrosis and fatty changes were investigated in these pancreatic tissues by 2 independent researchers in blind way. Serum fucosylated haptoglobin (Fuc-Hpt) levels in patients with chronic pancreatitis and pancreatic cancer were measured with lectin-antibody ELISA, using Aleuria aurantia lectin (AAL) and Pholiota squarrosa lectin (PhoSL). AAL recognizes all types of fucosylation and PhoSL recognizes core fucosylation. (Results) Pancreatectomy specimens showed a higher ratio of positive change in fibrosis (86% vs. 42%), fatty degeneration (72% vs. 44%), and inflammatory cell infiltration (14% vs. 3%) than control samples. Multivariate analyses demonstrated that each histological change was a significant, independent determinant for PDAC. In contrast, AAL-reactive Fuc-Hpt levels were increased in patients with pancreatic cancer and AAL-reactive Fuc-Hpt was though to be produced in the liver metastasis of pancreatic cancer. In contrast, PhoSL-reactive Fuc-Hpt levels were increased in patients with chronic pancreatitis, compared to pancreatic cancer. Correlations of PhoSL-reactive Fuc-Hpt levels and other biochemical parameters such as serum amylase, lipase, and elastase levels were not observed. The investigation of mechanisms underlying an increase of serum PhoSL-reactive Fuc-Hpt in patients with chronic pancreatitis is underway. (Conclusion) Cryptogenic pancreatitis might be a premalignant disease for pancreatic cancer and dramatic changes in fucosylation linkage on Fuc-Hpt were observed in pancreatic carcinogenesis. These findings give us a possibility of preventing or diagnosing pancreatic cancer at earlier stage, chronic pancreatitis. Citation Format: Eiji Miyoshi, Tomohiro Maekawa, Makiko Ueda, Mayuka Shimomura, Shinji Takamatsu, Kotarosumitomo Nakayama, Yoshihiro Kamada, Yasuhiko Tomita. A strategy for detecting high-risk groups for pancreatic cancer with glycol-biomarkers. [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 3122.

Abstract 2684: Clinical significance of GDP-mannose-4,6-dehydratase mutation and loss of fucosylation in colorectal cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC (Background and Aim) Oligosaccharides are one of the most important factors in the posttranslational modification of proteins and lipids. Glycomics, the systematic study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the post-genomics and post-proteomics era. In particular, fucosylation is one of the most important glycosylation involved in cancer and inflammation. Fucosylation, which comprises the transfer of a fucose residue to oligosaccharides and proteins, is regulated by many kinds of molecules, including several kinds of fucosyltranferases, GDP-fucose synthetic enzymes, and GDP-fucose transporter(s). Fucosylation is increased during carcinogenesis of many cancers through the up-regulation of expression of fucosylation-regulatory genes as described above. In contrast, we found a colon cancer cell line HCT116, which had complete loss of fucosylation due to the mutation of a GDP-fucose synthetic enzyme, GMDS (GDP-mannose-4, 6-dehydratase) a few years ago. While knockout mice of a GDP-fucose synthetic enzyme are lethal (Smith et al J Cell Biol, 158, 2002), HCT116 cells showed more aggressive pheno-type, including in vivo tumor growth and metastasis. The underlying mechanisms were found to be an escape from TRAIL-mediated immuno-surveillance of NK cells (Moriwaki et al, Gastroenterology 137, 2009). In the present study, we investigated whether or not the GMDS mutation was really found in clinical samples of colorectal cancer. (Subjects and Methods) Thirteen cases of liver metastasis, 6 cases of lymph node metastasis and 81 cases of the original cancer tissues derived from patients with colorectal cancer were included in this study. All projects were approved by the ethical committee of Osaka University. GMDS mutation was analyzed by RT-PCR using primers, which include all exons of GMDS gene. Immunohistochemical experiments were performed using AAL lectin, which recognizes all types of fucosylation. (Results and Discussion) Approximately 10-15% of GMDS mutation was found in the original cancer tissue as well as their metastatic liver cancer and lymph node metastasis. However no mutation was found in normal colon tissue around cancer. Most GMDS mutation was a hetero-type, but not a homo-type, which was found in HCT 116 cells. Immunohistochemical analysis showed complete loss of fucosylation in a few cases of metastatic cancer. These data indicate the involvement of GMDS mutation in the progression of colorectal cancer. Citation Format: Kotarosumitomo Nakayama, Kenta Moriwaki, Mayuka Shimomura, Hironobu Fujii, Shinji Takamatsu, Yoshihiro Kamada, Kohei Murata, Eiji Miyoshi. Clinical significance of GDP-mannose-4,6-dehydratase mutation and loss of fucosylation in colorectal 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 2684. doi:10.1158/1538-7445.AM2013-2684