Seven tumor‐associated autoantibodies as a serum biomarker for primary screening of early‐stage non‐small cell lung cancer

Abstract Objective The purpose of this study was to analyze the levels of tumor‐associated autoantibodies (TAAbs) in lung diseases and determine their diagnostic efficiency in early‐stage non‐small cell lung cancer (NSCLC). Methods We retrospectively analyzed the levels of 7‐TAAbs in 177 newly diagnosed early‐stage NSCLC patients, 202 patients with lung benign diseases and 137 healthy cases. The levels of a panel of 7‐TAAbs, including p53, GAGE7, PGP9.5, CAGE, MAGE A1, SOX2, GBU4‐5, were measured by ELISA. Results The serum levels of p53, GAGE7, PGP9.5, CAGE, MAGE A1, SOX2, and GBU4‐5 were not statistically different among NSCLC, benign and healthy groups (p > 0.05). The area under the curve (AUC) of 7‐TAAbs was all lower than 0.70. The sensitivity of combined detection was the highest (23.73%), while the specificity was the lowest (88.79%). The positive rates of PGP9.5, SOX2, and combined detection were significantly different among the three groups (p < 0.05). Among them, PGP9.5 and combined detection were significantly different between the NSCLC and benign groups (p < 0.05), PGP9.5, SOX2 and combined detection were significantly different between the NSCLC and healthy groups (p < 0.05). Conclusions The diagnostic efficiency of 7‐TAAbs in early‐stage NSCLC was not high, so it cannot be used alone as a screening method for NSCLC.

also high, and it reached 96.4% in NLST study. 6 With the popularization of LDCT, more micro pulmonary nodules have been found by LDCT. In the clinic, LDCT and clinical factors are considered in making a preliminary judgment and a risk rating of pulmonary nodules.
However, it is difficult to judge the nature of micro-nodules accurately based only on imaging and clinical features. 7 Histopathology, as an invasive mean, is used to identify the properties of these suspicious nodules. According to the guidelines for the management of incidental pulmonary nodules developed by Fleischner Society 2017, CT follow-up is usually used for some low-risk nodules due to the unsuitability of invasive methods when considering their very low probability of malignancy. 8 However, these low-risk nodules still have a malignant potential, and some patients feel anxious due to this uncertainty. Therefore, it is particularly meaningful to search non-invasive and high diagnostic efficiency indexes for early-stage LC screening, not only to avoid delayed diagnosis of malignant LC, but also to reduce the anxiety of patients with benign pulmonary nodules.
Tumor-associated autoantibodies (TAAbs) are produced by the immune system in response to peptides expressed at the surface of tumor cells or proteins that are released by tumors. 9 TAAbs have been frequently used in the diagnosis of malignancies, such as colorectal cancer, esophageal cancer, and LC. [10][11][12] However, scholars have different conclusions on their diagnostic efficiency. Some scholars considered that the incidence of autoantibody reaction against tumor-associated antigen (TAA) was very low, and these TAAbs should be needed to combine with protein array to improve their predictive value as tumor biomarkers. 13 At present, TAAbs combined with LDCT have been used in LC screening in some medical institutions. In DU Q study, who chose the same TAAbs panel as ours, he showed that the 7-TAAbs could distinguish between benign and malignant patients, with the sensitivity of 56.53% and the specificity of 91.60% in the LC diagnosis. 14 Similarly, our hospital has carried out 7-TAAbs test for a period of time to assist in LC screening. Unfortunately, we found that the TAAbs panel was not ideal for the initial LC screening. Therefore, we retrospectively analyzed the TAAbs data to explore the diagnostic value in LC, so as to provide a reference for clinical practice.
In our study, we selected a panel of 7-TAAbs, including p53, GAGE7, PGP9.5, CAGE, MAGE A1, SOX2, and GBU4-5, for the diagnosis of early-stage NSCLC. We compared the levels of 7-TAAbs in NSCLC, benign lung diseases and healthy cases, and explored its diagnostic efficiency in early-stage NSCLC.

TA B L E 1
The clinical features of the NSCLC group was carried out strictly according to the manufacturer's instructions.
In our study, the 7-TAAbs panel included p53, GAGE 7, PGP 9.5, it was judged as negative.

| Statistical analysis
The SPSS software version 25.0 was used for statistical analysis.

| Comparative analysis of the levels of 7-TAAbs among the NSCLC, benign and healthy groups
We compared the serum levels of p53, PGP9.5, SOX2, GAGE7, GBU4-5, MAGE A1, and CAGE in NSCLC, benign and healthy control groups. The serum levels of the 7-TAAbs were not statistically significant (p > 0.05) among the three groups ( Figure 1).

| Diagnostic efficiency of 7-TAAbs in the NSCLC group
The NSCLC group was defined as the disease group, and the be-  Table 2).

| The positive rate of 7-TAAbs in three groups
We compared the positive rate of a single marker and combined markers in the NSCLC, benign, and healthy groups. The positive rate of combined detection was higher than that of any single detection in the three groups. However, the positive rate was not high in NSCLC group with less than 50.00%. In addition, we compared the positive rate of 7-TAAbs among the three groups. PGP9.5, SOX2 and combined detection were significantly different among the three groups (p < 0.05). PGP9.5 and combined detection were significantly different between the NSCLC and benign groups (p < 0.05). PGP9.5, SOX2, and combined detection were significantly different between the NSCLC and healthy groups (p < 0.05). There were no statistical differences between the benign and healthy groups (p > 0.05), ( Figure 3).

| DISCUSS ION
Lung cancer screening methods include LDCT, X-ray, histopathology, and serological detection. With the improvement of health awareness and the popularity of physical examination, LDCT has been extensively used in clinical LC screening because of high sensitivity. 16 However, LDCT also had high false-positive rate.
Many patients were diagnosed with pulmonary nodules by LDCT, and some people were troubled because of the uncertain nature of these nodules. Histopathology is an invasive examination that is used for the diagnosis of suspected patients. Generally, it is not suitable for all patients who have pulmonary nodules to diagnose.
Early detection and treatment can prolong the survival of LC patients. Therefore, it is meaningful to find non-invasive laboratory indicators that have high diagnostic efficiency. The laboratory method can easily be popularized in the clinic, which is conducive to LC early diagnosis, and reduces the anxiety of some patients diagnosed with pulmonary nodules.
Tumor associated antigens (TAAs) which are highly expressed in the process of tumorigenesis and progression, can induce immune response. 17 Compared with TAAs, TAAbs have amplification effects and are easily detectable, thus TAAbs can be considered as tumor markers. 13 In recent years, TAAbs have been used in LC auxiliary diagnosis, however, the conclusion of their diagnostic efficiency was partially consistent. 11,18 This may be related to the enrolled patients and the selected panel of TAAbs. In the study of LI P, he showed that the levels of some TAAbs increased with the increase of pathological stages. 19 In our study, the NSCLC group was mainly the ad-

F I G U R E 2
The ROC curve of 7-TAAbs in NSCLC diagnosis. The NSCLC group was defined as the disease group, and the benign and healthy groups were defined as the control group.  p is the comparison among the NSCLC, benign and healthy groups. p1 is the comparison between the NSCLC and benign groups. p2 is the comparison between the NSCLC and healthy groups. p3 is the comparison between the benign and healthy groups. *p < 0.05 was statistically different among the three groups. NSCLC, non-small cell lung cancer only 13.0%, and the specificity was 88.9%. In our study, although the sensitivity was higher than theirs, it was still at a low level, indicating that the panel of 7-TAAbs was not sensitive enough in the diagnosis of early LC. In Li Y study, he found that the levels of TAAbs was closely related to the progress of the tumor, and TAAbs could be used as an indicator of curative effect monitoring and tumor recurrence in lung adenocarcinoma patients. 24  Non-invasive, easy to be carried out and high diagnostic efficiency projects were urgently needed in clinic, but there is a long way to go.

| CON CLUS IONS
The diagnostic efficiency of 7-TAAbs in early-stage NSCLC was not high, so it cannot be used alone as a screening method for LC. In some medical institutions that carried out TAAbs, the results should be carefully analyzed.

ACK N OWLED G EM ENTS
The authors would like to express their gratitude to EditSprings (https://www.edits prings.com/) for the expert linguistic services provided.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

AUTH O R CO NTR I B UTI O N S
LW collected the clinical data and divided then into different groups according to the inclusion criteria. CP analyzed the clinical data of the NSCLC, lung benign disease and normal control. CTT was a major contributor in writing the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets analyzed during the current study are available from the corresponding author on reasonable request.