Seromic profiling of colorectal cancer patients with novel glycopeptide microarray

Abstract

Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant posttranslational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures.Colorectal cancer develops in a multistep process that arises from genetic or epigenetic alterations.1 Most colorectal cancers can be treated by removal of early malignant lesions, 2,3 but despite this colorectal cancer remains the second most common cause of cancer-related death in the western world. 4 Current screening techniques, which include fecal occult blood, sigmoid and colonoscopy and computed tomographic colonography, are complicated with low compliance and high cost.5 Therefore, there is a need for biomarkers, which can identify colorectal cancer at early stages and aid in the surveillance and identification of high-risk populations.Broad genomic and proteomic approaches for identification of biomarkers have so far failed to develop simple, reliable and noninvasive screening test for early detection of colorectal cancer.6 Current serum assays detecting cancer glycoproteins such as CEA and CA-19-9 have limited use for early stages with low specificity and sensitivity. 7,8 As an alternative, circulating autoantibodies elicited by exposure to aberrant cancer proteins lacking immunological tolerance are emerging as promising biomarkers for the early detection of cancer.9-13 However, relatively few autoantibody epitopes have been identified and characterized to date.14 In a small study, we recently demonstrated the existence of such cancerassociated autoantibodies to the aberrantly O-glycosylated MUC1 mucin in patients with breast, ovarian and prostate cancer at time of diagnosis.15 These results provide basis for further discovery of autoantibody targets with the aim to increase sensitivity and organ specificity of biomarker assays through signatures of autoantibo...

Microvesicle Cargo of Tumor-Associated MUC1 to Dendritic Cells Allows Cross-presentation and Specific Carbohydrate Processing

“…In carcinomas that express MUC1, the tumor-associated glycoforms of MUC1 have been shown to be immune targets in vivo (27). In patients with cancer, CD8 þ T cells directed against MUC1 peptide epitopes were detected before and after vaccination (28)(29)(30), and the IgG auto-antibodies against specific tumor-associated glycoforms were found in patients with cancer with early-stage disease (5,31). Early work showed that MUC1 following internalization is blocked in the endolysosomal compartment of dendritic cells (9), suggesting that only a tolerizing CD4 þ T-cell response could be generated against this glycoantigen.…”

Colorectal Cancer Circulating Biomarkers

“…Importantly, a protein microarray technology is still being developed, and improvements are reported annually (Zhu and Snyder 2001;Liotta et al 2003;Ramachandran et al 2004;Chatterjee et al 2006;Kung and Snyder 2006;Oishi et al 2006;Hudson et al 2007;Ran et al 2008;Babel et al 2009;OrenesPinero et al 2010;Babel et al 2011;Pedersen et al 2011;Takulapalli et al 2012;Festa et al 2013). From the first commercially available human protein microarray consisting of 5,000 proteins in 2006, the number of proteins contained in the microarrays has grown up to 9,500 human full-length recombinant proteins in only one chip.…”

Glycome as Biomarkers

“…This study showed that the amount of autoantibodies against Globo H in breast cancer patients was significantly higher than normal blood donors. Pedersen et al (2011) generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The seromic profiling of immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various MUC1 and MUC4 glycopeptides carrying aberrant glycosylation.…”