Study On Gastric Cancer And Lung Cancer-specific IgG Fc N-glycosylation Modification Markers

Cancer is the second severest threat to human health only next to cardiovascular diseases.Gastric cancer and lung cancer are top-ranking in cancer-related morbidity and mortality.A cancer patient who is misdiagnosed as benign disease will miss the best time to intervene and has a lower chance to survival.Therefore,it will be of great importance to differentiate malignancy from benign diseases.Immunoglobulin G(IgG)is one of the most important immune moleculars in blood.It is well-known that glycosylation at Asn297 of Fc can modulate the functional interaction between IgG Fc fragment and its receptors,initiating the pro-or anti-inflammatory activities of antibodies,which has been proved to be associated with human physiological and pathological states.Nevertheless,previous studies only focused on total IgG,ignoring the effect of antigen-specific IgG.As the second most abundant protein in peripheral blood,not all IgG is associated with diseases.It has been proved that antigen-specific IgG and total IgG show differences in glycosylation between patients and healthy subjects.Therefore,changes of antigen-specific IgG maybe more accurately reflect the states of disease.This study operated native-polyacrylamide gel electrophoresis(Native-PAGE)to separate serum immunoinflammation-related protein complexes(IIRPCs),which were significantly elevated in disease states.IgG separated from IIRPCs ought to be disease-specific IgG(DSIgG).After separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),heavy chains of DSIgG were digested with trypsin.The enrichment of glycopeptides was performed using a natural material,poplar catkin.DSIgG glycopeptides attached to Fc region at the site of Asn297 were analyzed using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry(MALDI-FTICR MS).The differences of glycopeptide intensities or ratios were statistically analyzed in order to find potential biomarkers for cancer diagnosis and monitoring.1.DSIgG Fc N-glycosylation as personalized biomarkers to differentiate gastric cancer from benign gastric diseasesIn this study,DSIgGs derived from serum IIRPCs were isolated from 1037 serum samples including 525 patients with benign gastric diseases(BGDs)and 512 patients with gastric cancer(GC).Eight kinds of DSlgG1 N-glycoforms(GOF,GOFN,G1,GIF,G1FN,G1S,G2F,and G2FS)and fourteen kinds of DSIgG2 N-glycoforms(GOF,GOFN,G1,G1N,G1F,G1FN,G1S,G1FS,G2,G2S,G2N,G2F,G2FN,and G2FS)were observed in the mass spectra.Statistical analysis indicated that there were significant differences in DSIgG1 N-glycoforms including G1,G1S,G2F,G1FN and DSIgG2 N-glycoforms including GOF,GOFN,G1N,G1FS,G2,G2F,G2FN,G2FS.Significant changes in glycosylation features from DSIgG were observed between two pathophysiological states.Sialylation and galactosylation,especially digalactosylation,decreased and bisecting N-acetylglucosamine(GlcNAc)increased in GC patients,compared with BGDs patients.Receiver operating characteristic(ROC)analysis showed that galactosylation ratio of G2FN/G1FN from DSIgG2 had an excellent capability to distinguish female BGDs patients from female GC patients over the age range of 20-79 years,with the sensitivity of 82.6%,the specificity of 82.6%,and the area under curve(AUC)of 0.872.For males,changes in DSIgG glycoforms were closely associated with age.Over the age range of 40 to 59,a combination of G2F/G1F and G2FN/G2N of DSIgG2 had good ability to differentiate male GC patients from male BGDs patients,with the AUC of 0.846.Meanwhile,over the age range of 60 to 79,a combination of GIFS and G2FN/G2N of DSIgG2 and G2F/G0F of DSIgGl showed a good ability to distinguish male GC patients from male BGDs patients,with the AUC of 0.777.These findings indicate that DSIgG Fc glycoforms could reflect the difference in pathophysiological states between GC and BGDs,which may be used as potential personalized biomarker for GC diagnosis.2.DSIgG Fc N-glycosylation as personalized biomarkers to differentiate non-small-cell lung cancer from benign lung diseasesIn this study,glycopeptides of DSIgG from 509 patients with benign lung diseases(BLDs)and 477 patients with non-small-cell lung cancer(NSCLC)were detected by MALDI-FTICR MS and ratios of glycopeptides were investigated as diagnostic markers to differentiate NSCLC patients from BLDs patients.Statistical analysis indicated that fucosylation and sialylation increased and galactosylation decreased in NSCLC patients,compared with BLDs patients.ROC analysis showed that,panel a,a combination of G1F/G1,G1FS/G1S of DSIgG2 and G1F/G1 of DSIgGl,exhibited an acceptable diagnostic accuracy to differentiate NSCLC patients from BLDs patients below 60 years old,with the AUC>0.76,the sensitivities of>87%,and the specificities of>61%.Meanwhile,panel b,a combination of G1FS/G1F,G2F/G2 and G1FS/G1S of DSIgG2,had an acceptable diagnostic ability to differentiate NSCLC patients from BLDs patients above 60 years old,with the AUC>0.78,the sensitivities of>91%,and the specificities of>54%.These findings indicate that DSIgG Fc glycosylation ratios are closely associated with pathophysiological states between NSCLC and BLDs,which may be a potential personalized biomarker for NSCLC diagnosis.3.Disease-specific IgG glycosylation as personalized biomarker for monitoring disease progression of non-small-cell lung cancer patientsDisease progression is inevitable in the majority of patients with NSCLC due to the acquired drug resistance.Early and accurate detection of disease progression is benefit for instructing drug therapy in order to improve survival and life quality of patients.DSIgG glycopeptides of 413 serial serum samples from 36 patients with NSCLC were detected by MALDI-FTICR MS in this study.Kaplan-Meier curves indicated that galactosylation(DSIgG1 G2F/G1F)and fucosylation(DSIgG2 G1F/G1)were significantly associated with progression-free survival of NSCLC patients.DSIgG glycopeptide ratios(G1F/G1,G1S/G1 from DSIgG1 and G1S/G1,G1FN/G1N,G1FN/G0FN from DSIgG2)were employed to predict disease progression.Statistical analysis indicated that the median of lead time of disease progression based on glycopeptide ratios were 29 relative to clinical imaging,with the interquartile range of 16 to 34.These results demonstrate that DSIgG glycosylation may be a helpful personalized biomarker for monitoring disease progression of NSCLC patients.