Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

Abstract

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Fortythree proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. Molecular & Cellular Proteomics 8:2382-2395, 2009.

Seromic profiling of ovarian and pancreatic cancer

“…Whereas some studies used undefined protein extracts from tumor lysates (11,14,(28)(29)(30), others focused on limited numbers of candidate tumor antigens (24,25). To our knowledge, only two studies have performed a large-scale profiling of autoantibodies related to cancer using the extensive panel of human antigens present on ProtoArrays, one study probing with 12 colorectal cancer sera and 8 control sera (2), and the other probing with 30 ovarian cancer sera and 30 healthy controls (9). The study in colorectal cancer (2) led to the defi- Table S2.…”

“…A total of 19/197 (10%) antigens immunogenic by seromics in ovarian cancer patients and 2/28 (7%) in pancreatic cancer patients have been previously identified by serological screening of cDNA expression libraries from various other cancer types (SEREX ; Table S3), thereby confirming their immunogenic potential. For example, antigens ANXA2 or DNAJB1 were previously found to elicit autoantibodies in non-small-cell lung cancer (Tables S4 and Tables S5). Additionally, one target of ovarian cancer sera (MAPKAPK3) was recently identified as an immunoreactive antigen in colorectal cancer in one of the only other studies that used a similar strategy with ProtoArrays in a smaller sample set (2).…”

“…Arrays can be used with high throughput to determine patterns of antigens recognized by autoantibodies during the course of diseases, such as autoimmunity or cancer (1)(2)(3)(4)(5)(6)(7)(8)(9), as well as a way to characterize the repertoire of serological responses in healthy individuals (10). Defined protein collections assembled from phage expression systems or purified from recombinant sources were used to detect antibody responses in the serum of ovarian (11,12), breast (13), colorectal (2), pancreatic (14), or lung (15,16) cancer patients, and some of these responses appear to have diagnostic significance. In addition, protein array tools are also useful to measure changes in antibody responses to vaccination or immunotherapies, although it has yet to be applied to the monitoring of cancer treatments (17,18).…”

An RGD Motif Present in Cadherin 17 Induces Integrin Activation and Tumor Growth

“…Recombinant domains 6 (WT and mutant) were produced in Escherichia coli and purified according to standard procedures (16).…”

In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway

“…More than 100 TAAs have been identified by these endogenous antitumor autoantibodies (EAAs), including 78-kDa glucose-regulated protein [GRP78, also known as binding Ig protein (BiP)], p53, carcinoembryonic acid (CEA), and mucin 1 (MUC1) (2). The use of these autoantibody signatures as biomarkers in the early detection of CRC has been proposed (3)(4)(5). Typically, EAAs have not had a significant effect on tumor elimination, most likely due to immune tolerance induction by the tumor (6,7).…”