Comparison of Antinuclear Autoantibodies Expression Between COPD and Lung Cancer Patients

Comparison of Autoantibodies Expression Be- and Patients. Abstract Objectives: Circulating antinuclear autoantibodies (ANAs) have been detected in lung cancer patients and chronic obstructive pulmonary disease (COPD) patients as well. The specific differences and significance of ANAs in COPD patients and lung cancer patients remain unclear. Methods: We investigated the expression of circulating ANAs and their various known antibody subtypes in lung cancer patients, COPD patients and healthy controls, and compared the differences between each group, also the differences between patients over 70 and under 70 of age in each group and the differences between different genders. ANAs were tested by indirect immunofluorescence (IFT) and western blot analysis, and case-control study was used to investigate the differences between groups of 90 cases of lung cancer; 68 cases of COPD and 30 cases controls who were gender and age-matched. Results: The results of the research showed that the positive rate of ANAs in lung cancer patients was significantly increased compared with COPD patients(p=0.004) and healthy controls(P=0.00). Statistical analysis did not reveal any correlations between the patients’ age and the ANA positive results either in lung cancer group or in COPD group. The differences between different genders were not statistically significant; too. The most common autoantibody subtype was anti-Ro-52. Conclusions: The positive rate of circulating ANAs was significantly higher in lung cancer patients than in COPD patients.


Introduction
Antinuclear antibodies (ANAs) are commonly used as an initial test to screen for systemic autoimmune diseases, and the activity of autoantibody had been verified in the sera of cancer patients [1,2].
Emerging evidence suggests that ANAs are connected with COPD and low BMI, but not with smoking and forced expiratory volume in 1s [3]. Autoimmune are not rare among patients with cancer [1], previous study has shown that autoimmunity plays an important role in the pathogenesis of COPD [4]. Many epidemiological studies have agreed that the presence of COPD is associated with the development of lung cancer, but little is known about its mechanism [5]. How to recognize the significance of ANAs elevation in clinical work is an important part of disease diagnosis and differential diagnosis.
We conducted a case-control study to further clarify the characteristics of ANAs between cancer and COPD patients. Forced spirometry (before and after bronchodilation) [6] measurements in accordance with international guidelines.
The reference values were those of the Chinese population [7].

Measures and Clinical Data
All selected patients underwent demographic and clinical data collection, collecting venous blood before PSG after a minimum of 8h of fasting. Information on thorough medical history, age, gender, medication history and prescription, comorbidities were recorded.
Patients in the COPD group were examined for pulmonary function.
All measurements were performed by an experienced technician who was not aware of the protocol on the morning of the visit.

Immunofluorescence (IFT)
After fasting for at least 8 hours, venous blood was collected before PSG and injected into the coagulation tubes immediately, and centrifuged for 10 min, speed 3000 RPM at 20 degrees centigrade. According to the manufacturer's recommendations for the detection of antinuclear reagents by IFT, commercially available kits (EUROIMMUN, Medizinische labordiagnostika AG, Lubeck, Germany) were used. In short, frozen sections of unfixed HEp-2 cells were incubated with serum samples at room temperature for 30 minutes. After washing with phosphate buffered saline / tween (0.2% PBS tween), sections were exposed to FITC labeled Goat anti human IgG for 30 minutes. Immunofluorescence was observed by two independent researchers using Zeiss immunofluorescence microscope. ANA initial serum dilution was 1 / 10; all samples were diluted to the end of IFT residue. In the negative control group, the slides were incubated with single buffer solution and with FITC labeled antibody respectively. High titer serum or positive serum was used as positive control.

Immunoblot
Immunoblot was used for specific ANA reactivities---Anti-

Statistical Analysis
The matching distributions of age and sex between the groups were estimated by calculation. All statistical analyses were performed using SPSS software package (version 17.0; SPSS, Chicago, Illinois, USA). Continuous variables between groups were compared using independent sample T test. Pearson chi-square analysis and Fisher exact test were used to compare the discrete variables between the two groups. When P < 0.05, the difference was considered statistically significant.   Table 3. The distributions of age characters of participating people in different groups are in Figure 2.

The comparison of Antinuclear Autoantibodies Expression between Each Group
The results for each group were illustrated in Table 4. In our study ANAs were much more common in lung cancer patients (P=0.00)and COPD patients (P=0.049) than in control individuals.
Further comparison revealed that circulating ANAs in lung cancer patients were significantly higher than those in COPD patients (P=0.004).    Table 4).

Expression of antinuclear autoantibodies in different ages
In lung cancer group, ANA titers were elevated in 31 patients

Discussion
Studies have shown that lung cancer is one of the leading causes of death worldwide. It is a non immunogenic cancer that is resistant to immunomonitoring [8]. Circulating autoantibodies appear more frequently in cancer patients than in non cancer patients [9]. The incidence of ANAs in various types of cancer in different age groups of both sexes had been reported [10]. Our results showed that the ANAs positive rate of lung cancer patients was higher than that of the general population, which was consistent with previous studies.
Chronic obstructive pulmonary disease (COPD) is now the third leading cause of death in the world [11]. And COPD has other important manifestations beyond the lungs, known as systemic effects. These factors include unwilled weight loss, skeletal muscle dysfunction, and an increased risk of cardiovascular disease, osteoporosis, and depression. Low-level chronic systemic inflammation is one of the potential mechanisms of these systemic effects [12]. COPD is a polymerous disease. Autoimmunity may responsible for the pathogenesis of COPD [13]. Lung carcinoma is the most common concurrent comorbidities which COPD patients frequently suffer from, [11] the presence of COPD and emphysema is related to the increased risk factor of lung carcinoma [14].
ANAs react with various nuclear autoantigens. Previous studies have shown that although ANA positive is highly sensitive to certain rheumatic diseases, the presence of ANAs is non-specific and can be involved in a number of non-rheumatic factors, including malignancies, environmental exposure, drugs and infections [15].
They constitute a group of heterogeneous autoantibodies. [16] There are multiple case reports of ANAs in patients with malignancies [16]. The work of Fernandez Madrid F etc showed autoimmunity is a significant feature of squamous cell carcinoma, and suggested that the molecular characteristics of nuclear antigens recognized by ANAs may help to find valuable markers to distinguish LSCC from HNSCC [17]. The group of Nunez B revealed that abnormal titers of circulating ANA occurred in between a quarter and a third of clinically stable COPD patients [18]. Some authors found COPD is a heterogeneous disease and associated with a higher prevalence of ANAs [19].
However, it has been reported that aging is related to autoimmunity, and the positive rate of ANA was higher in the elder [20,21] [25] and the cDNA of human Ro52 was cloned three years later [26,27] In humans, the size of the Ro52 gene is 8.8 KB which stand on chromosome [11].
Ro52 is an interferon(IFN)-inducible protein [28] which is also induced by type I IFN-induced viral infection or toll-like receptor (TLR) binding [29]. Studies have shown that Ro52 is a negative regulator of proinflammatory cytokine production. And anti-Ro antibodies were frequently found to be involved in SLE, [30] SS/ SLE overlap syndrome, SCLE, and Neonatal lupus erythematosus (NLE) [31] NLE is a passively conveyed autoimmune disease that occurs in mothers of some infants with anti-Ro and / or anti-La antibodies [32] whose most common complication is congenital complete heart block (CCHB), which occurs in 0-5% of such pregnancies [33]. Anti-ro52 antibody is highly specific in primary biliary cirrhosis, an autoimmune liver disease [34] Although the causative role of autoantibodies in autoimmune diseases is not yet clear, it has been hypothesized that anti-Ro antibody might play a direct role in tissue damage. Anti-ro52 antibody may has pathological effects by directly damaging tissues and inhibiting the activity of Ro52 antigens. Our results revealed in lung cancer patients a higher frequency of anti-Ro-52, followed by anti-SSA. Our study has limitations, we could not assess associations with specific autoimmune diseases or other diseases, and the small sample sizes of some subgroups may limit our ability to detect differences in ANA prevalence on certain factors.
In summary, our results show that a higher frequency of elevated positive results of ANAs was observed in COPD than in normal people, however, the positive rate of circulating ANAs in lung cancer patients was significantly higher than in COPD patients.
Further study will help to understand the pathogenesis of COPD and lung cancer and the relationship between the two kind of diseases. The development of autoantibodies is the result of the destruction of human immune balance, [35] and we believe that the main role of the intratumor immune response can promote our understanding of tumor evolution and has important implications in clinical practice [36].

Conflict of Interest
None.