Comparative study of autoantibody responses between lung adenocarcinoma and benign pulmonary nodules

Introduction: The reduction in lung cancer mortality associated with computed tomography (CT) screening has led to its increased use and a concomitant increase in the detection of benign pulmonary nodules. Many individuals found to have benign nodules undergo unnecessary, costly, and invasive procedures. Therefore, there is a need for companion diagnostics that stratify individuals with pulmonary nodules into high-risk or low-risk groups. Lung cancers can trigger host immune responses and elicit antibodies against tumor antigens. The identification of these autoantibodies (AAbs) and their corresponding antigens may expand our knowledge of cancer immunity, leading to early diagnosis or even benefiting immunotherapy. Previous studies were performed mostly in the context of comparing cancers and healthy (smoker) controls. We have performed one of the first studies to understand humoral immune response in patients with cancer, patients with benign nodules, and healthy smokers. Methods: We first profiled seroreactivity to 10,000 fulllength human proteins in 40 patients with early-stage lung cancer and 40 smoker controls by using nucleic acid programmable protein arrays to identify candidate cancerspecific AAbs. Enzyme-linked immunosorbent assays of promising candidates were performed on 137 patients with lung cancer and 127 smoker controls, as well as on 170 subjects with benign pulmonary nodules. Results: From protein microarray screening experiments using a discovery set of 40 patients and 40 smoker controls, 17 antigens showing higher reactivity in lung cancer cases relative to the controls were subsequently selected for evaluation in a large sample set (n = 264) by using enzyme-linked immunosorbent assay. A five-AAb classifier (tetratricopeptide repeat domain 14 [TTC14], B-Raf protooncogene, serine/threonine kinase [BRAF], actin like 6B [ACTL6B], MORC family CW-Type zinc finger 2 [MORC2], and cancer/testis antigen 1B [CTAG1B]) that can differentiate lung cancers from smoker controls with a sensitivity of 30% at 89% specificity was developed. We further tested AAb responses in subjects with CT-positive benign nodules (n = 170), and developed a five-AAb panel (keratin 8, type II, TTC14, Kruppel-like factor 8, BRAF, and tousled like kinase 1) with a sensitivity of 30% at 88% specificity. Interestingly, messenger RNA levels of six AAb targets (TTC14, BRAF, MORC family CW-Type zinc finger 2, cancer/testis antigen 1B, keratin 8, type II, and tousled like kinase 1) were also found to increase in lung adenocarcinoma tissues based on The Cancer Genome Atlas data set. Conclusion: We discovered AAbs associated with lung adenocaricnoma that have the potential to differentiate cancer from CT-positive benign diseases. We believe that these antibodies warrant future validation using a larger sample set and/or longitudinal samples individually or as a panel. They could potentially be part of companion molecular diagnostic modalities that will benefit subjects undergoing CT screening for lung cancer.