The relation of the level of serum anti-TF, -Tn and -alpha-Gal IgG to survival in gastrointestinal cancer patients.

OBJECTIVE: To evaluate the relation of the level of serum anti-TF, -Tn and -αGal carbohydrate antibodies to survival in gastrointestinal cancer patients. METHODS: The level of anti-TF (Thomsen-Friedenreich antigen), -Tn and -αGal IgG was analysed in the serum of patients with gastric (n = 83) and colorectal (n = 51) cancers in the long-term follow-up, using ELISA with polyacrylamide glycoconjugates. To evaluate overall survival and the risk of death, the Kaplan-Meier method and the Cox proportional hazards model were used in the univariate analysis of patients groups. RESULTS: A significantly better survival was observed: (1) in patients with an increased level of anti-TF antibodies (all, stage III, T2-4, N1-2 and G3; P = 0.004-0.038, HR = 0.16-0.46); and (2) in patients with an increased level of anti-Tn antibodies (G1-2 tumors; P = 0.034-0.042, HR = 0.34-0.47). A significantly worse survival was observed in gastrointestinal, gastric and colorectal groups with an increased level of serum anti-αGal antibodies. This association depended on the patho-morphology of tumors (all, stages I-II, III, T2-4, N0, N1-2 and G1-2; P = 0.006-0.048, HR = 1.99-2.33). In the combined assessment of the anti-TF and -αGal antibodies level of the whole gastrointestinal group (n = 53), P = 0.002, HR = 0.25, 95% CI 0.094-0.655. In the follow-up, the survival time was shorter in patients whose level of anti-αGal antibodies rose (P = 0.009-0.040, HR = 2.18-4.27). The level of anti-TF antibodies inversely correlated with neutrophil/lymphocyte ratio (NLR, r = - 0.401, P = 0.004, n = 49). Patients with a higher level of anti-αGal antibodies and NLR values demonstrated a significantly worse survival (P = 0.009, HR = 2.98, n = 48). CONCLUSIONS: The preoperative levels of anti-TF, -Tn and -αGal antibodies and their dynamics are of prognostic significance. The method for the determination of circulating anti-carbohydrate antibodies may be a useful supplement in clinical outcome assessment.

Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival.

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.

A genetic variant of immunoglobulin gamma2 is strongly associated with immunity to mucin 1 in patients with breast cancer.

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes-genetic markers of IgG heavy chains and kappa-type light chains, respectively-contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin gamma2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.

The Thomsen-Friedenreich antigen and alphaGal-specific human IgG glycoforms: concanavalin A reactivity and relation to survival of cancer patients.

Glycan structures of IgG strongly influence the affinity for Fcgamma receptors and antibody effector functions. However, no particular attention has been paid yet to the glycosylation of tumor antigen-specific IgG. The objectives of this study were (i) to investigate the concanavalin A lectin (ConA) reactivity of human anti-Thomsen-Friedenreich (TF) and anti-alphaGal specific IgG in gastric cancer patients and healthy controls and (ii) to evaluate whether the ConA-reactivity of anti-TF and anti-alphaGal specific IgG is associated with the survival rate of patients with cancer. Total IgG was purified from the sera of patients with gastric cancer and healthy blood donors. The anti-TF and anti-alphaGal glycotope specific IgG were detected with ELISA using synthetic saccharide-polyacrylamide conjugates as antigen. In parallel plate, the ConA reactivity of the anti-TF or anti-alphaGal IgG was determined and the ConA index was calculated. Results show that serum anti-TF specific IgG antibodies of patients with cancer contain significantly higher content of ConA positive IgG glycoform compared to IgG of controls. No correlation between the ConA reactivity of anti-TF IgG and anti-alphaGal IgG was observed. High level of anti-TF IgG ConA reactivity was associated with a significantly lower survival rate of patients with gastric cancer.

The Thomsen-Friedenreich Antigen-Specific Antibody Signatures in Patients with Breast Cancer.

Alterations in the glycosylation of serum total immunoglobulins show these antibodies to have a diagnostic potential for cancer but the disease-related Abs to the tumor-associated antigens, including glycans, have still poorly been investigated in this respect. We analysed serum samples from patients with breast carcinoma (n = 196) and controls (n = 64) for the level of Thomsen-Friedenreich (TF) antigen-specific antibody isotypes, their sialylation, interrelationships, and the avidity by using ELISA with the synthetic TF-polyacrylamide conjugate as an antigen and the sialic acid-specific Sambucus nigra agglutinin (SNA) and ammonium thiocyanate as a chaotrope. An increased sialylation of IgG and IgM, but a lower SNA reactivity of IgA TF antibodies, and a higher level and avidity of the TF-specific IgA were found in cancer patients. Other cancer-related signatures were the highly significant increase of the IgG/IgA ratio and the very low SNA/IgA index in cancer, including patients with an early stage of the disease. These changes showed a good diagnostic potential with about 80% accuracy. Thus, the level of naturally occurring anti-TF antigen antibodies, their sialylation profile, isotype distribution, and avidity displayed cancer-specific changes that could serve as novel noninvasive Ab-based biomarkers for early breast cancer.

Hidden IgG Antibodies to the Tumor-Associated Thomsen-Friedenreich Antigen in Gastric Cancer Patients: Lectin Reactivity, Avidity, and Clinical Relevance.

Natural antibodies to the tumor-associated Thomsen-Friedenreich antigen (TF) are related to tumor immunosurveillance and cancer patients' survival. Hidden IgG antibodies (HAbs) to TF, their lectin reactivity, avidity, and clinical relevance were studied. HAbs were present in cancer patients and controls. A decreased level of IgG HAbs was detected in cancer. The HAbs level positively correlated with the sialospecific SNA lectin binding in purified total IgG (tIgG) in donors and cancer patients, indicating that HAbs are higher sialylated. The avidity of anti-TF IgG in tIgG samples was lower in cancer patients (P = 0.025) while no difference in the avidity of free anti-TF IgG was established. A negative correlation between the avidity of anti-TF IgG in tIgG and SNA binding in both groups was observed (P < 0.0001). The HAbs level negatively correlated with the anti-TF IgG avidity in tIgG only in donors (P = 0.003). Changes in the level of HAbs and Abs avidity showed a rather good stage- and gender-dependent diagnostic accuracy. Cancer patients with a lower anti-TF IgG avidity in tIgG showed a benefit in survival. Thus the TF-specific HAbs represent a particular subset of anti-TF IgG that differ from free serum anti-TF IgG in SNA reactivity, avidity, diagnostic potential, and relation to survival.

Increased Avidity of the Sambucus nigra Lectin-Reactive Antibodies to the Thomsen-Friedenreich Antigen as a Potential Biomarker for Gastric Cancer.

AIM: To determine whether the naturally occurring Thomsen-Friedenreich (TF) antigen-specific antibodies differ in avidity between cancer patients and controls to find a novel biomarker for stomach cancer. METHODS: Serum samples were taken from patients with cancer and controls. The level of TF-specific antibodies and their sialylation were determined using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA). The avidity was determined using ammonium thiocyanate as a chaotrope. RESULTS: A significantly higher SNA lectin binding to anti-TF antibodies was found in cancer patients irrespective of disease stage. The avidity of only IgM TF-specific antibodies was significantly higher in cancer patients compared to controls. The SNA-positive anti-TF antibodies of cancer patients showed a significantly higher avidity, P < 0.001. The sensitivity and specificity of this increase for gastric cancer were 73.53% and 73.08%, respectively, with a 73.2% diagnostic accuracy. The higher avidity of SNA-reactive anti-TF antibodies was associated with a benefit in survival of stage 3 cancer patients. CONCLUSION: The SNA-reactive TF-specific antibodies display a significantly higher avidity in gastric cancer patients compared to controls, which can be used as a potential serologic biomarker for gastric cancer. It appears that IgM is the main target responsible for the above changes.

Increased sialylation of anti-Thomsen-Friedenreich antigen (CD176) antibodies in patients with gastric cancer: a diagnostic and prognostic potential.

AIM: To study whether alterations in the sialylation of antibodies (Ab) specific to the Thomsen-Friedenreich (TF) glycotope have a diagnostic and prognostic potential in gastric cancer. METHODS: Serum samples were taken from patients with gastric carcinoma (n = 142) and controls (n = 61). The level of TF-specific antibodies and their sialylation was detected using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA). RESULTS: The level of TF-specific IgM was significantly decreased in cancer compared with controls (P ≤ 0.001). Cancer patients showed a higher level of SNA binding to anti-TF IgM and IgA (P ≤ 0.001) irrespective of disease stage, tumor morphology, and gender. Changes in the SNA/Ab index demonstrated moderate sensitivity (66-71%) and specificity (60-73%) for stomach cancer. The best diagnostic accuracy (100%) was achieved in 29% patients with high SNA binding and low anti-TF IgM level. This subset of patients demonstrated the poorest survival. CONCLUSION: Our findings are the first evidence that the increased sialylation of TF-specific Abs combined with a low level of anti-TF IgM is strongly linked to gastric cancer and patients survival, which can be used as a novel biomarker for cancer detection and prognosis.

Aberrant glycosylation of the anti-Thomsen-Friedenreich glycotope immunoglobulin G in gastric cancer patients.

AIM: To study whether alterations in the glycosylation of immunoglobulin G (IgG) specific to the Thomsen-Friedenreich glycotope (TF) have diagnostic and prognostic potential in gastric cancer. METHODS: Serum samples were obtained from patients with histologically verified gastric carcinoma (n = 89), healthy blood donors (n = 40), and patients with benign stomach diseases (n = 22). The lectin-enzyme-linked immunosorbent assay-based glycoprofiling of TF-specific IgG (anti-TF IgG) was performed using synthetic TF-polyacrylamide conjugate as antigen, total IgG purified by affinity chromatography on protein G sepharose, and lectins of various sugar specificities: mannose-specific concanavalin A (ConA), fucose-specific Aleuria aurantia lectin (AAL) and sialic acid-specific Sambucus nigra agglutinin (SNA). The sensitivity and specificity of the differences between cancer patients and controls were evaluated by receiver operator characteristic (ROC) curve analysis. Overall survival was analyzed by the Kaplan-Meier method. Time-dependent ROC curve statistics were applied to determine cut-off values for survival analysis. All calculations and comparisons were performed using the GraphPad Prism 5 and SPSS 15.0 software. RESULTS: The level of TF-specific IgG was significantly increased in cancer patients compared with non-cancer controls (P < 0.001). This increase was pronounced mostly in stage 1 of the disease. Cancer patients showed a higher level of ConA binding to anti-TF-IgG (P < 0.05) and a very low level of SNA lectin binding (P = 0.0001). No appreciable stage-dependency of the binding of any lectin to anti-TF IgG was found. A strong positive correlation between the binding of AAL and SNA was found in all groups studied (r = 0.71-0.72; P < 0.0001). The changes in ConA reactivity were not related to those of the fucose- or sialic acid-specific lectin. Changes in the SNA binding index and the ConA/SNA binding ratio demonstrated good sensitivity and specificity for stomach cancer: sensitivity 78.79% (95%CI: 61.09-91.02) and 72.73% (95%CI: 57.21-85.04); specificity 79.17 (95%CI: 65.01-89.53) and 88.64% (95%CI: 71.8-96.6), for the SNA binding index and the ConA/SNA binding ratio, respectively. The other combinations of lectins did not improve the accuracy of the assay. The low level of ConA-positive anti-TF IgG was associated with a survival benefit in cancer patients (HR = 1.56; 95%CI: 0.78-3.09; P = 0.19), especially in stages 3-4 of the disease (HR = 2.17; 95%CI: 0.98-4.79; P = 0.048). A significantly better survival rate was found in all cancer patients with a low reactivity of anti-TF IgG to the fucose-specific AAL lectin (HR = 2.39; 95%CI: 1.0-5.7; P = 0.038). CONCLUSION: The changes in the TF-specific IgG glycosylation pattern can be used as a biomarker for stomach cancer detection, and to predict patient survival.

Immunoglobulin allotypes influence antibody responses to mucin 1 in patients with gastric cancer.

There are significant interindividual differences in naturally occurring antibody responses to the tumor-associated antigen mucin 1 (MUC1), but the host genetic factors that might contribute to these differences have not been identified. The aim of the present investigation was to determine whether the variation in naturally occurring antibody levels to MUC1 in patients with gastric cancer is associated with GM and KM allotypes, genetic markers of IgG heavy chains and kappa-type light chains, respectively. A total of 169 Caucasian subjects with gastric cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. GM 3 23 5,13 phenotype was highly significantly associated with MUC1 IgG levels; subjects with this phenotype had lower antibody levels compared with those lacking this phenotype (median IgG level 65.5 relative units versus 91.0 relative units, P = 0.0058). In addition, this phenotype had an interactive effect with KM phenotypes on the levels of IgG antibodies to this antigen (P = 0.0081). Levels of MUC1 IgM antibodies were not associated with these genetic markers. These results show, for the first time, that GM and KM allotypes contribute to the interindividual differences in humoral immunity to MUC1.

Better survival of Helicobacter pylori infected patients with early gastric cancer is related to a higher level of Thomsen-Friedenreich antigen-specific antibodies.

The survival of patients with histologically verified gastric carcinoma at stage I (n = 44) and stage II (n = 43) was analysed by the Kaplan-Meier method depending on H. pylori serological status and a level of IgG and IgM antibody to tumor-associated Thomson-Friedenreich antigen (T Ag). In cancer patients at stage I, significantly better survival for H. pylori seropositive patients was observed compared to H. pylori seronegative patients (median SE survival time: 60.0 +/- 3.8 mths and 37.0 +/- 7.8 mths, respectively; P < 0.0004, log-rank test). Patients with higher level of T Ag-specific IgG antibody (strong responders) showed significantly and dramatically better (P < 0.00001) survival rate than weak responders. However, an association of better survival with a higher level of anti-T antibody level was limited to the H. pylori seropositive patients exclusively (P < 0.00001) with no difference for H. pylori seronegative group of patients. The level of IgM anti-T Ag antibody was not significantly related to the survival of patients at both stages of the disease, though better survival was noted in H. pylori seropositive IgM strong responders at approximately 40-60 months of observation. Statistically insignificant associations between survival and H. pylori status or anti-T antibody levels were also observed in a group of gastric cancer patients at stage II. In summary, the survival of patients with early gastric cancer (stage I) is significantly better in H. pylori seropositive patients, and this phenomenon may be in part explained by up-regulation of T Ag-specific IgG immune response in H. pylori infected individuals.

Expression of tumor-associated Thomsen-Friedenreich antigen (T Ag) in Helicobacter pylori and modulation of T Ag specific immune response in infected individuals.

We tested the hypothesis that the gastric cancer associated bacteria, Helicobacter pylori (H. pylori) express the cancer-related Thomsen-Friedenreich (T) antigen. We also analysed whether infection with H. pylori alters the amount of natural anti-T antibodies in the patients' sera. Cell surface membrane extracts of H. pylori NCTC 11637 strain and clinical isolates of H. pylori (n = 13) were analysed by immunoblotting and cell-ELISA with five different T antigen-specific monoclonal antibodies (MAbs). Two major protein bands of approximately 68 kDa and 58 kDa were immunostained on blots of H. pylori extracts with T specific MAbs but not immunostained with unrelated MAb. The specificity was shown in that immunostaining was blocked with peanut agglutinin (PNA) and rabbit antiserum to T antigen. The binding of T specific MAb to the 58 kDa protein band was also blocked by rabbit antiserum against heat shock proteins of H. pylori. The relative expression of T antigen-related proteins differed among H. pylori strains, with 68 kD associated T antigen expression higher in patients with more severe pathology. The level of IgG antibody to T epitope in patients with gastric cancer (n = 66) and normal blood donors (n = 62) were compared and the level of anti-T Ab in gastric cancer patients was significantly lower than that in normal blood donors. A significant positive correlation between T specific antibody in serum and H. pylori IgG antibody level was found in H. pylori-infected normal blood donors (P < 0.001), but this correlation was not found in H. pylori-infected cancer patients. In summary, the cancer related T epitope is expressed in H. pylori and modulation of T antigen-specific immune response in H. pylori-infected individuals suggests that H. pylori infection may alter natural immune mechanisms against cancer.