Identification of fucosylated haptoglobin-producing cells in pancreatic cancer tissue and its molecular mechanism.

Fucosylated haptoglobin is a well-established glyco-biomarker of pancreatic cancer. We recently established a novel anti-glycan antibody (10-7G mAb) that specifically recognizes fucosylated haptoglobins, including prohaptoglobin (proHpt). Serum concentrations of the 10-7G value, as measured by ELISA, were increased in patients with pancreatic cancer relative to the healthy controls. However, it is currently unknown which specific tissue or cell type produces fucosylated haptoglobins or proHpt. In the present study, we performed immunohistochemical (IHC) and ELISA analyses of pancreatic cancer tissue samples using 10-7G mAb. Among 21 pancreatic tissue sections, only 1 showed direct staining of pancreatic cells with the 10-7G mAb. However, 12 of the 21 sections stained positively for immune cells. Although there was no significant difference in the 10-7G expression between the positive and negative staining IHC groups, the median value of serum 10-7G was slightly higher in IHC-positive cases. Among many assayed leukemic cell lines, differentiated THP-1 cells (a human acute monocytic leukemia cell line) were found to have the highest levels of proHpt, per Western blot using 10-7G mAb. Interestingly, production of proHpt in vitro was dramatically increased under either hypoxic conditions or after IL-6 treatment. These results suggest that immune cells, including macrophages, in the pancreatic tissue microenvironment produce fucosylated haptoglobin and proHpt. Thus, fucosylated haptoglobins can be detected by the 10-7G mAb and may be a promising biomarker for pancreatic cancer.

Detection of fucosylated haptoglobin using the 10-7G antibody as a biomarker for evaluating endoscopic remission in ulcerative colitis.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammation of the digestive tract. Although fecal and serum biomarkers have been extremely important and supportive for monitoring of IBD, their low sensitivity and high variability characteristics limit clinical efficacy. Thus, the establishment of better biomarkers is expected. Fucosylation is one of the most important glycosylation modifications of proteins. Fucosylated haptoglobin (Fuc-Hpt) is used as a biomarker for several cancers and inflammation-related diseases. We recently established a novel glycan monoclonal antibody (mAb), designated 10-7G, which recognizes Fuc-Hpt. We developed an enzyme-linked immunosorbent assay (ELISA) to measure serum levels of Fuc-Hpt (10-7G values). AIM: To investigate the usefulness of the serum 10-7G values as a potential biomarker for monitoring disease activity in IBD. METHODS: This was a case control study. Intestinal tissues of IBD patients (n = 10) were examined immunohistochemically using the 10-7G mAb. We determined 10-7G values using serum from patients with ulcerative colitis (UC, n = 110), Crohn's disease (n = 45), acute enteritis (AE, n = 11), and healthy volunteers (HVs) who exhibited normal (n = 20) or high (n = 79) C-reactive protein (CRP) levels at medical check-up. We investigated the correlation between the 10-7G value and various clinical parameters of IBD patients by correlation analysis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the usefulness of the 10-7G values as a biomarker for clinical and endoscopic remission of UC compared to conventional serum biomarkers. RESULTS: In the immunohistochemical analysis, positive 10-7G mAb staining was observed in lymphocytes infiltrating into inflammatory sites of the mucosal layer and lymphoid follicles. The 10-7G values were significantly higher in patients with IBD (P < 0.001) and AE (P < 0.05) compared with HVs. In addition, 10-7G values were correlated with clinical examination parameters related to inflammation in patients with UC, particularly the CRP level (rs = 0.525, P = 0.003) and clinical activity index score (rs = 0.435, P = 0.038). However, there was no correlation between 10-7G values and CRP in HVs with high CRP levels, suggesting that the 10-7G values is not the same as a general inflammation biomarker. ROC curve analysis showed that area under the curve (AUC) value of 10-7G values for the diagnosis of endoscopic remission was higher than other biomarkers (AUC value = 0.699). CONCLUSION: The serum 10-7G value is a novel biomarker for evaluating intestinal inflammation and endoscopic mucosal healing in UC.

Identification of the epitope of 10-7G glycan antibody to recognize cancer-associated haptoglobin.

We previously identified fucosylated haptoglobin (Fuc-Hpt) as a clinical serum biomarker of pancreatic cancer and established the novel glycan monoclonal antibody (mAb) 10-7G. This antibody recognizes cancer-associated haptoglobin including Fuc-Hpt and the precursor of haptoglobin. Interestingly, Western blot analysis showed that the 10-7G mAb reacts with the haptoglobin α chain, which has no N-glycan potential sites; haptoglobin ß chain has four N-glycan sites. In this study, we identified the epitope for the 10-7G mAb using haptoglobin deletion mutants, as well as inhibition ELISA with recombinant peptides. We illustrated molecular graphics to show a relationship between the epitope and the ß chain. Furthermore, we hypothesized that the 10-7G mAb minimally recognizes normal haptoglobin, but aberrant glycosylation on the ß chain causes conformational changes, enabling the 10-7G mAb to easily access the epitope within the α chain. Because 10-7G values, an enzyme-linked immunosorbent assay-immobilized 10-7G mAb, in patients with pancreatic cancer varied by haptoglobin phenotype, the amount of aberrant glycosylation needed to affect haptoglobin conformation probably depends on haptoglobin phenotype. Taken together, the 10-7G mAb recognized characteristic peptides on the haptoglobin α chain as a result of conformational changes and is a promising tool for diagnosing pancreatic cancer by haptoglobin phenotype.