Carcinoembryonic Antigen: A Predictor of Inammatory Condition in Patients with Allergic Bronchopulmonary Aspergillosis?

Background: Allergic bronchopulmonary aspergillus (ABPA) is a complex non-infectious pulmonary benign disease caused by immune response against to aspergillus. Carcinoembryonic antigen (CEA) is a tumor marker but also elevated in some benign diseases. Few studies on ABPA with elevated serum CEA levels have been reported. Objects: This study aims to comb the clinical characters of ABPA with elevated serum CEA. Methods: 20 patients diagnosed as ABPA were divided into two groups (CEA normal and CEA elevated) for retrospective analysis. The eosinophil count and ratio, IgE level in the pretherapy and post-treatment were compared. Serum samples of patients with ABPA (n = 20) and asthma (n = 20), healthy controls (n=20) were collected. Levels of cytokines (IL-4, IL-5, GM-CSF, IFN-γ) were analyzed by enzyme-linked immunosorbent assay. Results: We found that in ABPA patients with normal serum CEA levels, eosinophil counts and IgE levels decreased more obviously after treatment. Besides, we established higher serum levels of IL-4, IL-5, GM-CSF and IFN-γ in ABPA patients with elevated serum CEA levels. Conclusion: For the ABPA patients with elevated serum CEA levels, CEA may serve as a monitoring indicator for severity and treatment ecacy of ABPA.


Introduction
Allergic bronchopulmonary aspergillus(ABPA) is a complicated disorder due to immune response to aspergillus adhering to the airway, characterized by central tracheal dilatation and repeated pulmonary in ltration 1 . The classic pathological characteristics of ABPA include mucoid impaction, eosinophilic pneumonia, bronchiolitis obliterans, granulomatous bronchiolitis and pulmonary brosis 2,3 . The patients with ABPA often present with fever, wheezing, cough, bronchial hyperreactivity, or hemoptysis 4 . Human studies and animal experiments have shown that Th2 cells and relevant cytokines play a critical role in the progression of ABPA 5 . It is well known that CEA is a broad-spectrum tumor marker, which usually elevated in a wild variety of tumors such as colorectal cancer, breast cancer and lung cancer 6 . An increased levels of CEA can be acquired by a blood test, which commonly used for diagnosis, recurrence or progression of malignant diseases 7 . Some benign diseases such as viral hepatitis, cirrhosis, pancreatitis and ulcerative colitis have also reported elevated serum CEA levels 8,9 . Rarely, ABPA has been described with elevated CEA.
In this study, we observed 20 patients with ABPA, their total blood IgE levels, peripheral blood eosinophil counts and ratios before and after treatment and baseline CEA levels were collected, then evaluate the changes. Furthermore, we also detected a series of relevant cytokines in partial of the patients, compared with health volunteers and asthma patients.

Study subjects and patients:
We retrospectively analyzed 20 patients diagnosed with ABPA from Xiangya Hospital of Central South University (each patient was only included once) from January 2017 to May 2020. The diagnosis of ABPA were in accordance with criteria of working group under the International Society for Human and Animal Mycology (ISHAM). Patients' data were collected including age, gender, blood routine, pulmonary function, imaging, bronchoscopy, IgE (total and speci c), serum CEA, CRP, ESR and clinical and medication records. Table 1 demonstrates the details of the ABPA patient groups. The diagnosis of asthma was based on the Global Initiative for Asthma (GINA) guidelines. Patients with serious illness, malignancy or other diseases with elevated serum CEA were excluded. Twenty healthy volunteers were recruited into the healthy control group. Serum of 9 ABPA patients were collected prior to treatment. All asthma samples were collected in our outpatient clinic before treatment. Levels of IL-4, IL-5, IFN-γ and GM-CSF in serum were analyzed by ELISA according to the manufacturer's instruction (All kits were purchased from eBioscience).

1.Clinical materials
As shown in Table 1, among 20 ABPA patients, 6 of them have signi cant elevated serum CEA levels above baseline and others were normal. Most patients clinically presented with wheeze, cough, dyspnea, chest tightness and hemoptysis. The lung function also indicated ventilatory dysfunction most. All the patients received glucocorticoid and antifungal treatments after diagnosed as ABPA. However, we found that most ABPA patients with elevated CEA presented with mucus plugs in bronchoscopy and manifestations of CT compare with ABPA patients with normal serum CEA. As we can see, The IgE levels, peripheral blood eosinophil counts and ratios did not differ in two groups. The erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also found no difference (Fig. 1). After a period of combination therapy of corticosteroid and antifungal agent treatments, the serum CEA levels were decrease than baseline levels. The levels of serum total IgE, peripheral blood eosinophil counts and ratios were both decreased in the two groups, while the degree of decline was more pronounces in the patients with normal serum CEA (Fig. 2).

2.cytokine Levels
We collected serum samples from the ABPA patients, asthma patients and healthy volunteers, and detected the concentrations of cytokines. We found that ABPA patients with elevated serum CEA produced more IL-4 and IL-5 than ABPA patients with normal serum CEA. In addition, production of IL-4 and IL-5 were signi cantly higher in ABPA patients with elevated serum CEA than in healthy subjects. Moreover, expression of IFN-γ and GM-CSF showed the similar trend in all groups (Fig. 3).

Discussion
ABPA was rst reported in 1952 by Hinson et al 10 . In the past, ABPA has been de ned as a rare disease in China 11 . Due to ABPA remains under recognized and under diagnosed, it is often misdiagnosed as asthma, tuberculosis, lung cancer and other diseases [12][13][14] . The typical features of laboratory investigations include increased total IgE level, serum speci c IgE levels and peripheral blood eosinophils. In this study, all patients with ABPA ful ll above diagnostic criteria.
CEA is a glycoprotein involved in cell adhesion that is usually produced during fetal development but stops before birth. Serum CEA usually elevated in many cancers including lung, breast, gastric and so on. High levels can also be found in some benign lung conditions. Ahmed Fahim et have demonstrated serum CEA correlates with disease severity in idiopathic pulmonary brosis(IPF) 15 20 . In accordance with our study in bronchoscopy and imaging manifestations, we found that mucus plugs were seen more frequently in ABPA patients with elevated serum CEA levels, which indicate that serum CEA levels may related with the prognosis of ABPA.
Sensitization to Aspergillus fumigatus is the rst step in pathogenesis of ABPA. Repeated exposure to aspergillus fumigatus causes immune response such as IgE elevation, Th2 cytokines high expression, eosinophils in ltration and airway remodeling 4 . Studies have shown that the number of Th2 cells increased in peripheral blood and BALF in patients with ABPA, which reinforce the function of interlukin-4(IL-4), further activate B cells and enhance IgE production, which promote disease activity and progression. The increase of IL-5 and GM-CSF will promote eosinophil recruitment, thus exacerbate airway damage 21 . In addition, Khosravi AR et al have found that airway epithelial cells exposed to aspergillus fumigatus spores produced more IFN-γ, which lead to brief periods of increased IFN-γ within the body to promote aspergillus clearance and play a defensive role 22,23 . In our study, ABPA patients with elevated serum CEA levels have a higher level of Th2 cytokines (IL-4 and IL-5),GM-CSF and IFN-γ,which indicate that a more intensely in ammatory response occurrence in vivo environment of patients. This reveal that serum CEA levels may related to degree in ammation and disease severity.
Currently, treatment e cacy of ABPA usually evaluated by monitoring pulmonary imaging and serum total IgE levels. There should be an improved radiographic lung changes and a 35% minimum reduction in total IgE levels 24,25 . Researchers have found that serum CEA levels could be reduced after treatment compared to baseline in ABPA patients 16 . The rare common characteristics of the six cases described in the paper is the extremely high serum CEA level, which decreased after treatment synchronizing with serum total IgE levels, suggesting that serum CEA level correlated with the progress in treatment of the disease.
In summary, ABPA patients with elevated CEA levels show a more severe in ammatory response and a poor response to treatment compare to ABPA patients with normal CEA. This indicates that CEA may serve as a monitoring indicator for severity and treatment e cacy of ABPA. Surely, there were limitations in our study. First, we collected blood samples of patients at a single point,and the sample data were incomplete. Second, the numbers of cases we included were relatively small. Our ndings were just a hint, in order to better evaluate the immunological characteristics of ABPA and the diagnostic value of serum CEA levels in ABPA, additional studies are required.

Conclusion
Our study found that CEA may be a predictor of in ammatory condition in patients with allergic bronchopulmonary aspergillosis. This will provide additional meaningful metrics both for clinical and scienti c examination.  Figure 1 Clinical and immunological data of ABPA patients (two groups: CEA normal(n=14) and CEA elevated(n=6)). Serum CEA levels (A); serum total IgE levels (B); peripheral eosinophil counts and ratios (C); serum ESR(E) and CRP(F) in ABPA. ABPA allergic bronchopulmonary aspergillosis, CEA Carcinoembryonic antigen.

Figure 2
Longitudinally observation of serum levels of CEA(A), peripheral blood eosinophil counts(B) and serum total IgE levels(C) in patients with ABPA (two groups CEA normal(n=14) and CEA elevated(n=6)). ABPA allergic bronchopulmonary aspergillosis, CEA Carcinoembryonic antigen.