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An important role for tumor infiltrating B lymphocytes (TIL-B) in the immune response to cancer is emerging; however, very little is known about the antigen specificity of antibodies produced in situ . The presence of IgA antibodies in the tumor microenvironment has been noted although their biological functions and clinical significance are unknown. This study used a 91-antigen microarray to examine the IgG and IgA autoantibody repertoires in breast cancer (BC). Tumor and adjacent breast tissue supernatants and plasma from BC patients together with normal breast tissue supernatants and plasma from healthy controls (patients undergoing mammary reduction and healthy blood donors) were analyzed to investigate relationships between autoantibodies and the clinical, histological and immunological features of tumors. Our data show that >84% of the BC samples tested contain autoantibodies to one or more antigens on the array, with ANKRD30BL, COPS4, and CTAG1B being most frequently reactive. Ex vivo TIL-B responses were uncoupled from systemic humoral responses in the majority of cases. A comparison of autoantibody frequencies in supernatants and plasma from patients and controls identified eight antigens that elicit BC-associated autoantibody responses. The overall prevalence of IgG and IgA autoantibodies was similar and while IgG and IgA responses were not linked they did correlate with distinct clinical, pathological and immunological features. Higher levels of ex vivo IgG responses to BC-associated antigens were associated with shorter recurrence-free survival (RFS), HER2 overexpression and lower tumor-infiltrating CD8 + T cell counts. Higher IgA levels were associated with estrogen and progesterone receptor-negative cancers but were not significantly associated with RFS. Furthermore, ex vivo IgA but not IgG autoantibodies reactive to BC-associated antigens were linked with germinal center and early memory B cell maturation and the presence of tertiary lymphoid structures suggesting that these TIL-B are activated in the tumor microenvironment. Overall, our results extend the current understanding of the antigen specificity, the biological and the clinical significance of IgG and IgA autoantibodies produced by BC TIL-B in situ .

Background: Serum autoantibodies against tumor-associated antigens (TAAs) are detectable in early-stage gastric cancer patients; however, the time point during cancerogenesis when they appear in circulation is still obscure. Methods: In this study, we developed a recombinant antigen microarray and analyzed the prevalence of autoantibodies against 102 TAAs in 829 gastric cancer patients and 929 healthy controls from Caucasian and Asian populations, as well as 100 patients with chronic atrophic gastritis and 775 individuals staged according to different grades of intestinal metaplasia. Results: Six antigens, including CTAG1B/CTAG2, DDX53, IGF2BP2, TP53, and MAGEA3, were predominantly reacting with sera from gastric cancer patients when compared with healthy controls, and the seroreactivity was associated with intestinal-type gastric cancer, but not with patients' Helicobacter pylori status, grade, age, gender, or stage of gastric cancer. We detected gastric cancer-associated seroreactivity in 13% of patients with advanced/severe intestinal metaplasia, which was increased in comparison with mild/moderate intestinal metaplasia (5.3%) and was comparable with that seen in early-stage gastric cancer patients (12%). Moreover, by testing serum samples taken 1 to 9 years before the clinical diagnosis of 18 incident gastric cancer cases, we detected autoantibody responses against several TAAs-SOX2, MYC, BIRC5, IGF2BP1, and MUC1. Conclusions: Our results suggest that humoral immune response against TAAs is generated already during premalignant stages. Impact: Based on the obtained results, cancer-associated autoantibodies might make a valuable contribution to the stratification of high-risk patients with premalignant lesions in the stomach through enhancing the positive predictive power of existing risk models. Cancer Epidemiol Biomarkers Prev; 26(10); 1564-74. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Autoantibodies against tumor-associated antigens (TAAs) have been shown to serve as highly specific serological biomarkers for the diagnosis of various solid cancers. Although the autoimmunity against thyroid tissue specific antigens has been studied extensively, so far, the autoantibody responses against common TAAs such as cancer-testis antigens (CTAs), mutated or differentiation antigens have not been comprehensively analyzed in patients with thyroid cancer., Objective: The current study aims to characterize the frequency of autoantibody responses against common TAAs in patients with thyroid cancer and benign thyroid nodules., Methods: A phage-displayed antigen microarray comprising 65 TAAs was produced and tested with sera from 53 patients with thyroid cancer, 90 patients with benign thyroid nodules and 96 cancer-free individuals, 100 melanoma, 54 breast cancer and 14 lung cancer patients as controls., Results: A panel of 6 TAAs was identified that preferentially reacted with sera from patients with thyroid cancer. The top ranked antigen in this panel was GAGE1 eliciting autoantibody response in 6% of patients with thyroid cancer but not with benign nodules, whereas no reactivity to other CTAs was detected in the sera from patients with thyroid cancer., Conclusions: Although six TAAs, including one CTA, showed thyroid cancer-associated reactivity, overall, spontaneous humoral immune responses against TAAs are rare in thyroid cancer and their utility for the development of non-invasive assay for the differential diagnosis of thyroid nodules is limited.

Autoantibodies against tumor-associated antigens are very attractive biomarkers for the development of noninvasive serological tests for the early detection of cancer because of their specificity and stability in the sera. In our study, we applied T7 phage display-based serological analysis of recombinant cDNA expression libraries technique to identify a representative set of antigens eliciting humoral responses in patients with gastric cancer (GC), produced phage-antigen microarrays and exploited them for the survey of autoantibody repertoire in patients with GC and inflammatory diseases. We developed procedures for data normalization and cutoff determination to define sero-positive signals and ranked them by the signal intensity and frequency of reactivity. To identify autoantibodies with the highest diagnostic value, a 1,150-feature microarray was tested with sera from 100 patients with GC and 100 cancer-free controls, and then the top-ranked 86 antigens were used for the production of focused array that was tested with an independent validation set comprising serum samples from 235 patients with GC, 154 patients with peptic ulcer and gastritis and 213 healthy controls. The receiver operating characteristic curve analysis showed that 45-autoantibody signature could discriminate GC and healthy controls with area under the curve (AUC) of 0.79 (59% sensitivity and 90% specificity), GC and peptic ulcer with AUC of 0.76 and GC and gastritis with AUC of 0.64. Moreover, it could detect early GC with equal sensitivity than advanced GC. Interestingly, the autoantibody production did not correlate with histological type, H. pylori status, grade, localization and size of the primary tumor, whereas it appeared to be associated with the metastatic disease., (Copyright © 2012 UICC.)

In the current study we attempted to evaluate the suitability of T7 Select 10-3b and lambdaKM8 phage display systems for the identification of antigens eliciting B cell responses in cancer patients and the production of phage-displayed antigen microarrays that could be exploited for the monitoring of autoantibody profiles. Members of 15 tumour-associated antigen (TAA) families were cloned into both phage display vectors and the TAA mini-libraries were immunoscreened with 22 melanoma patients' sera resulting in the detection of reactivity against members of 5 antigen families in both systems, yet with variable sensitivity. T7 phage display system showed greater sensitivity for the detection of antibodies against members of CTAG, MAGEA and GAGE families, both systems showed equal performance in detecting the reactivity against MAGEC and SSX2 while only lambdaKM8 allowed the detection of anti-CTAGE5 antibodies. The biological properties of both phages turned out to be equally suitable for the production of antigen microarrays however in line with the plaque assay the sensitivity for the detection of various autoantibodies differed between the vectors. However, presumably due to the higher variability of the background signals in the microarray assay, it turned out to have comparable, in some cases even slightly lower sensitivity than the plaque assay. Next, we explored the repertoire of antigens that could be identified by screening T7 phage-displayed testis cDNA library with sera from melanoma patients. From the 243 antigens identified, only 24 represented known genes translated in their natural reading frame and included known TAAs like Annexin XI-A and a novel potential CT antigen SPAG8. Another 12 were uncharacterised genes but the remaining clones contained DNA fragments in non-natural reading frames that most likely represent mimotopes, nevertheless, they may turn out to be valid biomarkers.

An important role for tumor infiltrating B lymphocytes (TIL-B) in the immune response to cancer is emerging; however, very little is known about the antigen specificity of antibodies produced in situ. The presence of IgA antibodies in the tumor microenvironment has been noted although their biological functions and clinical significance are unknown. This study used a 91-antigen microarray to examine the IgG and IgA autoantibody repertoires in breast cancer (BC). Tumor and adjacent breast tissue supernatants and plasma from BC patients together with normal breast tissue supernatants and plasma from healthy controls (patients undergoing mammary reduction and healthy blood donors) were analyzed to investigate relationships between autoantibodies and the clinical, histological and immunological features of tumors. Our data show that >84% of the BC samples tested contain autoantibodies to one or more antigens on the array, with ANKRD30BL, COPS4, and CTAG1B being most frequently reactive. Ex vivo TIL-B responses were uncoupled from systemic humoral responses in the majority of cases. A comparison of autoantibody frequencies in supernatants and plasma from patients and controls identified eight antigens that elicit BC-associated autoantibody responses. The overall prevalence of IgG and IgA autoantibodies was similar and while IgG and IgA responses were not linked they did correlate with distinct clinical, pathological and immunological features. Higher levels of ex vivo IgG responses to BC-associated antigens were associated with shorter recurrence-free survival (RFS), HER2 overexpression and lower tumor-infiltrating CD8+ T cell counts. Higher IgA levels were associated with estrogen and progesterone receptor-negative cancers but were not significantly associated with RFS. Furthermore, ex vivo IgA but not IgG autoantibodies reactive to BC-associated antigens were linked with germinal center and early memory B cell maturation and the presence of tertiary lymphoid structures suggesting that these TIL-B are activated in the tumor microenvironment. Overall, our results extend the current understanding of the antigen specificity, the biological and the clinical significance of IgG and IgA autoantibodies produced by BC TIL-B in situ.