The Diagnostic And Pathologic Significance Of Abnormally Glycosylated IgG

Every year, roughly7.2to7.5million people worldwide die from cancer,55%of whichoccur in developing countries. The survival of cancer is highly dependent on the pathologicalstage upon diagnosis, early detection of cancer greatly increases five-year survival rate.However, current tumor markers have poor sensitivity and specificity or they are not elevatedearly enough in the cancer process to be useful for screening. Newer markers are needed toovercome these problems and allow diagnosis of cancer at an earlier stage. Glycosylations areactively involved in tumor development and progression. Immunoglobulin G is among themajor glycoproteins in circulation. We proposed that IgG glycan variants could be exploitedfor biomarker development aimed at diagnosing early-stage cancer, and IgG glycan variantscould functionally modulate IgG.This study aims to assess the diagnostic value of IgG N-glycans in large sample clinicalcase control study for hepatocellular carcinoma and colorectal cancer, and to examine whetherIgG glycan variants could functionally modulate IgG.Part1: N-glycans of Serum IgG: New Diagnostic and Prognostic Markers forHepatitis B Virus-related Hepatocellular CarcinomaThe objective of this study was to examine N-glycans of IgG in HBV-relatedhepatocellulcar carcinoma(HCC), and to determine whether N-glycans of IgG could be used asdiagnostic and prognostic markers for HBV-related HCC. A total of387cases of HBV-relatedhepatocellulcar carcinoma, liver cirrhosis, HBV carriers and healthy controls were segregatedinto training set (n=150) and4validation sets (n=237). Serum core-α-1,6-fucosylated IgG wasquantified by lectin blot and immunoturbidimetry after protein G/A or Lens culinarisagglutinin chromatography. Serum IgG N-glycan profiles were analyzed with DNAsequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). Diagnosticperformances of serum IgG N-glycans were assessed in training set and further validated infour validation cohorts. The prognostic performances were assessed in a36-month follow upset (n=55). IgG in cultured supernatants of four liver cancer cell lines was determined byELISA. We found that the percentage of core-α-1,6-fucosylated IgG (LCA-IgG/IgG×100%)demonstrated better diagnostic performances (accuracy84.4-85.8%, sensitivity90.0-91.7%) inthe training set compared to alpha-fetoprotein(accuracy63.3-72.5%, sensitivity～65.0%).Independent validations further confirmed the improved diagnostic efficacies of LCA-IgG/IgG,especially in discriminating hepatocellulcar carcinoma from liver cirrhosis. Increased LCA-IgG/IgG and abundance of agalacto core-α-1,6-fucosylated biantennary glycan ofIgG(peak1) corresponded to worse survival in a36-month follow up study. Invitroobservations revealed hepatoma cell lines could secrete IgG. All these results confirmed thatLCA-IgG/IgG is a promising noninvasive marker and valuable supplement to AFP andcore-fucosylated AFP (AFP-L3) for HCC identification. Both the abundance of the agalactocore-α-1,6-fucosylated biantennary glycan of IgG (Peak1) and the LCA-IgG/IgG displayfavourable effects in HCC prognostic management.Part2: serum IgG N-glycans marker for colorectal cancer diagnosisThis study aims to identify the N-glycan variations of IgG and to assess the diagnosticvalues of IgG N-glycan marker in colorectal cancer (CRC). A total of228subjects includingCRC, adenoma and healthy controls were collected. N-glycan profiling of serum IgG wasanalyzed by DSA-FACE. Serum core-α-1,6-fucosylated IgG was quantified byimmunoturbidimetry after Lens culinaris agglutinin chromatography. IgG N-glycan markerswere identified based on DSA-FACE and the receiver operating characteristic curve analysis.The result showed that peaks1,2and7in CRC group were higher than those in the controlgroups (p<0.05). The level of total core fucose residues increased in CRC and adenomasignificantly. The area under the ROC (AUC) of Log(P1+P2) were0.703. Compared withroutine clinical tumor markers CEA, the sensitivity and the accuracy of Log(P1+P2) wasimproved28%and7%respectively. Log(P1+P2) decreased significantly after radical surgery.Furthermore, Log(P1+P2) was negatively correlated with survival time (P<0.05). Thus weconcluded that IgG N-glycans Log(P1+P2) could be a potential biomarker for colorectal cancerdiagnosis.Part3: Glycan variants could functionally modulate cetuximabGlycosylations are actively involved in tumor development and progression.Immunoglobulin G is among the major glycoproteins in circulation. This study aims toexamine whether IgG N-glycan variants could occur in diseases and functionally modulateIgG.A total of377cases of HBV-related liver disease (n=150) and colorectal disease (n=227)was collected. Serum IgG N-glycan profile was analyzed by DSA-FACE. The results showedthat the abundance of bisecting Nacetylglucosamine (GlcNAc) residues of IgG was increasedsignificantly in LC and CRC, but almost similar among the other diseases.To evaluate the functional modulation of IgG glycan variants on IgG, the gene encodingthe Chinese hamster glycosylation enzyme β,1–4-N-acetylglucosaminyltransferase III (GnT-III) was cloned and coexpressed in a recombinant production Chinese hamster ovary(CHO) cell line expressing a chimeric mouse/human anti-EGFR IgG1antibody(cetuximab).DSA-FACE analysis showed the enzyme had added bisecting-GlcNAc residues in some(7.16%to41.39%) of the N-linked oligosaccharides isolated from cetuximabs expressed bythese cell lines. The binding capacity of Fab to EGFR-positive cell was measured by cellproliferation assay. ADCC against EGFR positive tumor cell line was evaluated using healthydonor peripheral blood mononuclear cells as effectors and lactate dehydrogenase release as amarker of cell killing. We found that cetuximab with higher bisecting N-acetylglucosamineresidues could improve the Fab cell-apoptosis potency to EGFR-positive cell and promote thekilling of EGFR-positive target cell at approximately2-fold higher than the parent.