Abstract
There is considerable evidence that the presence of cancer can elicit a humoral immune response to specific proteins in the host, and these resulting autoantibodies may have potential as noninvasive biomarkers. To characterize the autoantibody repertoire present in the sera of patients with lung adenocarcinoma, we developed a high-density peptide microarray derived from biopanning a lung cancer phage display library. Using a 2,304-element microarray, we interrogated a total of 250 sera from Michigan lung cancer patients and noncancer controls to develop an "autoantibody profile" of lung adenocarcinoma. A set of 22 discriminating peptides derived from a training set of 125 serum samples from lung adenocarcinoma patients and control subjects was found to predict cancer status with 85% sensitivity and 86% specificity in an independent test set of 125 sera. Sequencing of the immunoreactive phage-peptide clones identified candidate humoral immune response targets in lung adenocarcinoma, including ubiquilin 1, a protein that regulates the degradation of several ubiquitin-dependent proteasome substrates. An independent validation set of 122 serum samples from Pittsburgh was examined using two overlapping clones of ubiquilin 1 that showed 0.79 and 0.74 of the area under the receiver operating characteristics curve, respectively. Significantly increased levels of both ubiquilin 1 mRNA and protein, as well as reduced levels of the phosphorylated form of this protein, were detected in lung tumors. Immunofluorescence using anti-ubiquilin 1 antibodies confirmed intracellular expression within tumors cells. These studies indicate that autoantibody profiles, as well as individual candidates, may be useful for the noninvasive detection of lung adenocarcinoma.
Highlights
Lung cancer is the leading cause of cancer-related deaths for both men and women in industrialized countries
Once in a microarray format, these enriched phage-peptide clones can be used to interrogate serum samples for humoral immune response markers. Using this 2.3K phage-peptide microarray, we evaluated sera from 150 lung adenocarcinoma patients and 100 noncancer control subjects (Supplementary Table S3)
Seven immunoreactive phage-peptides clones of www.aacrjournals.org ubiquilin 1 spanned 112 amino acids, from aa478 to aa589, and two clones spanned 125aa, from aa465 to aa589 (Fig. 2A). Both peptide stretches of ubiquilin 1 were the target of autoantibodies in lung adenocarcinoma patients relative to control subjects (P < 0.0001; Fig. 2B)
Summary
Lung cancer is the leading cause of cancer-related deaths for both men and women in industrialized countries. We combine phage display technology with protein microarrays to develop a powerful platform to identify and characterize an autoantibody signature for lung adenocarcinoma patients that can be evaluated, in multiplex, to develop diagnostic biomarkers. An independent cohort of sera, including 62 lung adenocarcinomas and 60 controls (Supplementary Table S4), matched for both age and smoking status and collected between 2000 and 2005 was provided by the University of Pittsburgh Cancer Institute/Hillman Cancer Center.
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