Anti-nuclear antibody (ANA) testing is useful for diagnosing systemic autoimmune rheumatic diseases. However, ANA can be detected in healthy individuals and patients with non-rheumatic diseases [1]. ANA positivity is frequently observed in various types of cancers. Several authors have suggested that ANAs are not only related to autoimmune diseases, but also to neoplastic diseases [2-4].

ANAs are autoantibodies against nuclear and cytoplasmic proteins of a cell. They are formed when the cellular contents are released into the extracellular environment during cell death. In neoplastic diseases, apoptosis or necrosis of tumor cells may cause ANA formation [5].

Although the presence of ANA in cancer is regarded as an epiphenomenon, some studies have indicated that autoantibodies may be used for early cancer detection and may play a role in prognosis [2, 4, 6]. Other studies have shown that the generation of autoantibodies in patients with malignancies may induce the development of autoimmune and rheumatic features [3, 7].

Breast cancer is the most common cancer in women. Previous reports have shown that the prevalence of ANA is higher in breast cancer patients than in patients with benign breast diseases or healthy controls, although the prevalence is variable, ranging from 35% to 99% [8-10]. Regarding immunofiuorescence patterns and titers, one study found that dense fine speckled patterns and fine/coarse speckled patterns were common in breast cancer patients, with only 20% of patients having a titer of 1:320 or higher [10]. Additionally, it has been suggested that ANA positivity could be used for early diagnosis, but it is not linked to other prognostic characteristics of breast cancer [2, 10, 11].

There is a lack of data on ANAs in breast cancer among Asian populations, and the clinical value of ANA positivity is not fully understood. In this study, we examined the prevalence of ANA in Asian patients with breast cancer and established an association between the presence of ANA and prognostic variables. We also investigated whether the presence of ANA was accompanied by the detection of other autoantibodies and the manifestation of rheumatism.

Patients who visited an outpatient clinic of Chungnam National University Hospital for breast masses between 2016 and 2019 and provided signed informed consent were enrolled in this study. Biopsies of breast masses were performed for diagnosis. A total of 187 newly diagnosed women (125 with breast cancer and 62 with benign breast diseases) were included, and none of them had a history of cancer or connective tissue disease. This study was approved by the institutional review board of our institution.

Venous blood samples were collected at the time of diagnosis and after surgery and centrifuged to separate serum. Serum aliquots were stored at -80℃ until ANA and extractable nuclear antigens (ENA) tests were performed. ANAs were detected using an indirect immunofiuorescence assay, Mosaic HEp-20-10 (Euroimmun, Lübeck, Germany) in a fully automated IF Sprinter analyzer (Euroimmun), according to the manufacturer’s instructions. The screening dilution was 1:80, and ANA titers were determined to 1:1,280 by successive two-fold dilutions. Samples were interpreted as ANA positive when a distinct fiuorescence pattern was identified according to the International Consensus on ANA Patterns (ICAP) nomenclature [12]. Samples with ANAs were analyzed for antibodies against ENA (SSA, SSB, centromere, RNP, Sm, Scl70, and Jo-1) and mitochondria using a line immunoassay, EUROLINE ANA Profile 3 (Euroimmun).

The clinical and histopathologic characteristics of patients with breast cancer were obtained from medical record reviews. Histopathological characteristics included stage, lymph node status, hormone receptor expression, human epithelial growth factor 2 (HER2) status, and p53 expression.

The prevalence, fluorescence patterns, and ANA titers were compared between the breast cancer and benign breast diseases groups. The clinical and histopathological characteristics of patients with breast cancer, were compared between the ANA-positive and ANA-negative groups. Clinicopathological parameters were analyzed using the Mann-Whitney U test or chi-square test. Statistical analyses were performed using MedCalc version 20.115 (MedCalc Software, Mariakerke, Belgium). A P value less than 0.05 was considered statistically significant.

The median age at diagnosis in patients with breast cancer and benign breast disease was 50 (23–72) years and 46 (24–77) years, respectively, and the median age was significantly higher in patients with breast cancer (P=0.02). The staging and histopathological characteristics of 125 breast cancer patients are summarized in Table 1. The stages of breast cancer were as follows: stage 0 (in situ), 40 patients; stage I, 42 patients; stage IIA, 12 patients; stage IIB, 28 patients; and stage IIIA, 3 patients.

Positive ANA test results were observed in 30 out of 125 (24.0%) patients with breast cancer and 5 out of 62 (8.1%) patients with benign breast disease. The prevalence of ANA positivity was significantly higher in the breast cancer group (P=0.01). The fiuorescence patterns and ANA titers in the breast cancer and benign breast disease groups are shown in Table 2. Speckled pattern, centromere pattern, cytoplasmic pattern, and mixed pattern, which refer to the presence of more than one ANA pattern, were only observed in the breast cancer group, but their frequencies were not statistically different from those in the benign breast disease group. In contrast, the frequencies of nucleolar pattern and dense fine speckled pattern were significantly higher in the benign breast disease group than in the breast cancer group (P=0.03 and P<0.01, respectively). Sixteen (53.3%) ANA-positive breast cancer patients exhibited a titer ≥1:160, but all ANA-positive patients with benign breast disease showed a titer of 1:80. The frequency of ANA titers ≥1:160 was significantly higher in the breast cancer group than in benign breast disease group (P=0.03).

The clinical and histopathological characteristics were compared between the ANA-positive and ANA-negative breast cancer groups (Table 3). The breast tissues with p53 immunostaining in at least 10% of the cell nuclei were defined to having p53 overexpression [10]. The frequencies of stage III, nodes N2/N3, hormone receptor positivity, HER2 positivity, and p53 overexpression did not differ between the two groups. During the median follow-up period of 55 months (range, 12–78), seven patients experienced recurrence, but the frequency was not related to ANA positivity.

Among breast cancer patients with ANA positivity, anti-ENA antibodies were only detected in four (13.3%) patients, and their ANA titers were 1:320 in one, 1:640 in one, and >1:1,280 in two patients. Anti-centromere antibody (ACA) was present in three patients showing a discrete speckled pattern, and anti-SSA/anti-SSB antibodies were found together in one patient showing a speckled pattern. In addition to anti-ENA antibodies, anti-mitochondrial antibodies were detected in three patients, showing speckled or cytoplasmic patterns, and their ANA titers were ≥1:320. None of the ANA-positive patients had autoantibodies included in the EUROLINE Profile 3 assay, except for anti-ENA and anti-mitochondrial antibodies. None of the patients with autoantibodies were diagnosed with rheumatic disease during the follow-up period.

For decades, researchers have reported that ANAs are frequently found in the sera of patients with malignancies [2-4, 6, 7, 10]. Previous studies showed that ANAs are detected in approximately 30% of patients with solid neoplasms of the breast, colon, and lungs, or with lymphoma [2-4]. Additionally, the prevalence of ANA positivity is higher in patients with chronic liver disease who develop hepatocellular carcinoma than in those with the most common antecedent clinical conditions such as liver cirrhosis or chronic hepatitis [13]. An overrepresentation of ANAs was confirmed and persisted in the cancer group with malignant pelvic masses compared to the benign group [6].

In the case of breast cancer, a recent study reported that the prevalence of ANA positivity was significantly higher in patients with breast cancer (44.4%) than in patients with benign breast diseases (15.7%) and healthy controls (5.4%) [10]. In this study, we consistently found that breast cancer patients had a higher prevalence of ANA positivity than patients with benign breast disease. Therefore, we confirmed that the frequent occurrence of ANA in the Asian population with breast cancer is a common finding that may be explained by an anti-tumor immune response. In our study, the prevalence of ANAs in breast cancer (24.0%) and benign breast disease groups (8.1%) was lower compared to the prevalence reported in other studies [8-10]. According to our previous data, in the Korean population where ANA testing is referred to as screening for systemic autoimmune rheumatic diseases, the prevalence of ANAs was lower than that in the Western population [14]. We predicted that the difference in prevalence observed in this study may be affected by different Hep-2 cell lines, subjective interpretations, and different ethnic groups.

In the aspect of immunofiuorescence patterns and titers, breast cancer patients and benign breast disease patients showed a significant difference. Common immunofiuorescence patterns in the cancer group were mixed, centromere, cytoplasmic, and speckled patterns, while dense fine speckled and nucleolar patterns were mainly seen in the benign breast diseases group. Different from a previous result [10], in the present study, dense fine speckled pattern was not common in breast cancer, and the prevalence of fine/coarse speckled pattern showed no significant difference between the two groups. We believe that immunofiuorescence patterns in breast cancer are not specific, and the detected autoantibodies determine the staining patterns. A previous review reported that variable staining patterns are observed in cancer owing to related autoantibodies [15].

Furthermore, we found that ANA titers ≥1:160 was only observed in the cancer group, as previously described [10]. The higher incidence of ANA titers ≥1:160 in breast cancer patients may suggest that the immune system is more strongly stimulated to nuclear antigens by tumor cell death. In particular, serum samples with detected autoantibodies (anti-centromere, anti-SSA/SSB, anti-mitochondria) showed ANA titers ≥1:320. This finding indicates that stronger immune stimuli induce the development of multiple autoantibodies. Although titers ≥1:80 are considered ANA positive, no autoantibodies were detected in breast cancer and benign breast disease patients with a low ANA titer (1:80). These findings show that a low ANA titer in breast cancer patients could be a part of the immune response, independent of the presence of autoantibodies, and may be disregarded.

Rheumatic paraneoplastic symptoms and syndromes are reported to be frequent in patients with cancer-related ANA positivity; however, molecular differences between autoantigens involved in cancer and those involved in systemic autoimmune rheumatic diseases are proposed [3]. In the present study, none of the patients with autoantibodies had concomitant or newly diagnosed rheumatic diseases during the follow-up period, and an association between the presence of ANA plus autoantibodies and systemic autoimmune rheumatic diseases was not demonstrated. Therefore, the presence of autoantibodies in breast cancer is more likely related to the cancer diagnosis than to rheumatic diseases.

ACAs are detected in various autoimmune diseases such as CREST syndrome, Sjögren’s syndrome, and primary biliary cirrhosis. Rattner et al. concluded that ACAs were found in a high proportion of patients with breast cancer because centromere proteins (CENP-F antigens) were identified in breast carcinoma tissue but were rarely observed in normal tissues [16]. An Asian population-based study of systemic sclerosis showed that ACA positivity needs to be considered as a significant cancer risk factor [17]. Our breast cancer patients with ACA had no history of systemic sclerosis and were not diagnosed during the follow-up period. Anti-mitochondrial antibodies are found in autoimmune liver and skin diseases, but have also been reported in patients with infiltrating ductal breast carcinoma and ductal carcinoma in situ [9]. Like ACA, the presence of anti-mitochondrial antibodies was not related to the development of autoimmune liver or skin diseases. Therefore, ACAs and anti-mitochondrial antibodies may be autoantibodies detected in breast cancer patients.

It is still not clear whether ANAs play a role in the prognosis of cancer patients. Some studies have shown that the presence of ANAs is related to a good tumor prognosis [4, 18, 19], and it has been proposed that a better prognosis may be caused by the anti-tumor activity of ANAs. However, other studies have not demonstrated a relationship between ANAs and prognosis [2, 10, 11], and contradictory data have been reported [6]. In the present study, we could not link the presence of ANAs to prognostic variables. This finding suggests that ANAs in breast cancer do not represent anti-tumor activity, but immunogenic stimulation by tumor cells.

In conclusion, we demonstrated that the prevalence of ANA positivity was higher, and titers ≥1:160 were more common in patients with breast cancer compared to those with benign breast diseases. The presence of ANAs was not associated with any tumor prognostic factors; however, the small number of subjects did not allow us to conclude a prognostic association. Therefore, further studies with larger sample sizes are warranted. The presence of ANA and additional autoantibodies may not be related to the development of rheumatic diseases.