Identification of 14-3-3𝛉 as an Antigen that Induces a Humoral Response in Lung Cancer

Abstract

We have implemented a strategy to identify tumor antigens that induce a humoral immune response in lung cancer based on the analysis of tumor cell proteins. Chromatographically fractionated protein extracts from three lung cancer cell lines were subjected to Western blotting and hybridization with individual sera to determine serum antibody binding. Two sets of sera were initially investigated. One set consisted of sera from 19 newly diagnosed subjects with lung adenocarcinoma and 19 matched controls. A second independent set consisted of sera from 26 newly diagnosed subjects with lung adenocarcinoma and 24 controls matched for age, gender, and smoking history. One protein that exhibited significant reactivity with both sets of cancer sera (P = 0.0008) was confidently identified by mass spectrometry as 14-3-3u. Remarkably, significant autoantibody reactivity against 14-3-3u was also observed in an analysis of a third set consisting of 18 prediagnostic lung cancer sera collected as part of the Beta-Carotene and Retinol Efficacy Trial cohort study, relative to 19 matched controls (P = 0.0042). A receiver operating characteristic curve constructed with a panel of three proteins consisting of 14-3-3u identified in this study, plus annexin 1 and protein gene product 9.5 proteins previously identified as associated with autoantibodies in lung cancer, gave a sensitivity of 55% at 95% specificity (area under the curve, 0.838) in discriminating lung cancer at the preclinical stage from matched controls.

Identification of a New Panel of Serum Autoantibodies Associated with the Presence of In situ Carcinoma of the Breast in Younger Women

“…However, new studies with larger cohorts need to be done across multiple populations before clinical development to show that CIS could be prospectively detected with the use of our five-autoantibody panel. Interestingly, Hanash et al has recently shown that autoantibody-signature cancer diagnosis is possible months before cancer reaches a size that can be picked up by imaging technology (7,50). Such investigations should be done for breast cancer.…”

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

“…Several approaches have been used to identify TAAs in cancer, including natural protein arrays prepared with fractions obtained from two-dimensional LC separations of tumoral samples (29,30) or protein extracts from cancer cells or tissue (9,31) probed by Western blot with patient sera, cancer tissue peptide libraries expressed as cDNA expression libraries for serological screening (serological analysis of recombinant cDNA expression libraries (SEREX)) (22,32), or peptides expressed on the surface of phages in combination with microarrays (17,18,33,34). However, these approaches suffer from several drawbacks.…”

Mass spectrometry of peptides and proteins from human blood

“…Major protein depletion, solution isoelectric focusing, and 1D PAGE resulted in 575 and 2,890 protein identifications from plasma and serum, respectively . Identifying the targets of immune reactions to proteins from cancer cells, normal self cells, and bacteria has been greatly facilitated by the use of PAGE followed by immunological probing with patient sera and mass spectrometry (Brichory et al, 2001;Hong et al, 2006;Pereira-Faca et al, 2007;Basagana et al, 2008;Bunk et al, 2008;Desmetz et al, 2008;Fujita et al, 2008;Hamrita et al, 2008;Nagashio et al, 2008;Taylor et al, 2008;Zhong et al, 2008;Kim et al, 2008b;Liu et al, 2008b;Forgber et al, 2009;He et al, 2009;Patwa et al, 2009;Rao et al, 2009;Shimada et al, 2009;Tamesa et al, 2009;Yi et al, 2009). A large number of protein isoforms in serum, or EDTA versus citrated plasma, were detected by fluorescent labeling prior to separation of the intact proteins by charge, hydrophobic character and molecular mass (Misek et al, 2005).…”

“…The peak height or integration method may well be applied on numerous MS instruments (Pereira-Faca et al, 2007;von Haehling et al, 2007;Jacot et al, 2008;Bereman, Williams, & Muddiman, 2009;Mas et al, 2009). It will be a good practice to add recovery and internal quantification standards into crude serum prior to a limited number of fractionation steps to control variation (Mirzaei et al, 2009).…”