Anti-DFS70 in Systemic Autoimmune Rheumatic Diseases: History
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Liudmila Zotova
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Victoria Kotova
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Zakhar Kuznetsov

The diagnosis of systemic autoimmune rheumatic disease (SARD) or its exclusion is carried out taking into account the results of immunological studies, primarily antinuclear antibodies (ANA) and specific autoantibodies. Often, during ANA analysis via indirect immunofluorescence reaction on cellular and tissue substrates, a dense fine speckled 70 (DFS70) fluorescence pattern is observed.

  • anti-DFS70
  • systemic autoimmune rheumatic disease
  • ANA

1. Introduction

Systemic autoimmune rheumatic diseases (SARDs) are characterized by polyclonal activation of B cells and the formation of a broad spectrum of specific autoantibodies, which in turn trigger immune-inflammatory damage to tissues and internal organs. The main diagnostic laboratory markers of frequently encountered autoimmune diseases, such as systemic lupus erythematosus (SLE), systemic scleroderma (SSc), Sjögren’s syndrome (SjS), polymyositis/dermatomyositis (PM/DM), and others, are antinuclear antibodies (ANA)—a heterogeneous group of autoantibodies directed to various components of the cell nucleus and cytoplasm. Due to the fact that modern diagnostic methods allow researchers to detect the autoantibodies to antigens located in various cell structures, including nuclear constituents, nuclear membrane, mitotic spindle apparatus, cytosol, cytoplasmic organelles, and cell membranes, a more accurate term for ANA is “anticellular antibodies” (anticell, AC), which is reflected in the modern nomenclature of immunofluorescence patterns recommended by the International Consensus on ANA Patterns—ICAP [1]. Positive results of the determination of ANA are among the diagnostic criteria for autoimmune diseases; they are utilized to assess disease activity, prognosis, and the characteristics of clinical-laboratory subtypes of the disease, and they serve as predictors of pathology development at the preclinical stage—all these factors underline the significance of this parameter in medicine. It is also important to mention the fact that the detection of autoantibodies may precede the clinical manifestation of the disease; for example, according to retrospective studies, elevated ANA levels were detected in the serum of 78% of systemic lupus erythematosus (SLE) patients up to 10 years prior to diagnosis [2][3].

2. Diagnostics of DFS70 Pattern

The DFS70 pattern and autoantibodies were initially described by Ochs R.L. et al. in 1994 [4], and its presence was described in patients with interstitial cystitis. This pattern is characterized by a heterogeneous dense fine speckled staining of the nucleoplasm of interphase cell nuclei and chromatin in the mitotic zone. The nuclear target antigen was named DFS70 based on the reactivity of the autoantibodies with a 70 kDa protein in Western blotting. Later, it was established that the DFS70 antigen is identical to a protein known as a transcriptional coactivator p75 or lens epithelium-derived growth factor, LEDGF [5], which has functions as a transcriptional coactivator p75 and a growth factor for lens epithelial cells; however, the use of the synonym LEDGF/p75 in routine practice is not entirely accurate, as the direct influence of the DFS70 antigen on lens development has not been established. Nevertheless, in scientific literature, there is an equivalence between the terms anti-DFS70, anti-LEDGF, and LEDGF/p75. Anti-DFS70 antibodies are primarily of the immunoglobulin of G class, but in certain atopic conditions, immunoglobulin of E class antibodies is also found.
To confirm the presence of antibodies to DFS70 in ANA-positive sera, methods such as solid-phase enzyme-linked immunosorbent assay, immunoblotting, chemiluminescent immunoassay, and HEp-2 IFA with selective antibody adsorption using a knockout DFS70/LEDGF cell line are currently used. A one-step analysis of DFS70 antibodies using the IFA method on HEp-2/DFS70 cells eliminates the need for additional confirmatory tests when investigating these antibodies, so using a substrate (HEp-2 ELITE/DFS70-KO) composed of a mixture of standard HEp-2 cells and genetically engineered DFS70-Ko HEp-2 cells that do not express the DFS70/LEDGF/p75 antigen, and this prevents the binding of DFS antibodies to the target antigen, allowing for a clear differentiation between DFS and classical types of nuclear staining.

3. Assessment of Detecting DFS70 Antibodies in Clinical Practice

It has been established that up to 20% of healthy individuals can be seropositive for ANA in HEp-2 IFA, which in turn, in half of the cases, is due to the presence of DFS70/LEDGF/p75 antibodies (pattern AC-2 according to the nomenclature of ANA patterns as agreed upon by the International Consensus on Patterns—ICAP) [6][7]. The relatively high rate of false-positive results for the ANA test (referring to situations where subsequent autoimmune diseases do not develop) among healthy people and patients with non-autoimmune diseases often raises concerns and alertness for both patients themselves and primary care physicians, creating an unnecessary burden on the healthcare system as it leads to the performance of additional, including expensive investigations.
Among healthy individuals, the frequency of detecting isolated anti-DFS70 antibodies (i.e., in the absence of other specific antibodies for SARDs autoantibodies) ranges from 2% to 21.6% (with an average of 6.8%), in ANA-positive donors, this frequency varies from 23.8% to 57% (with an average of 43.9%) [5][7][8][9]. Furthermore, these antibodies are often found at high titers (frequently reaching levels of 1:5120). However, it is worth noting a considerable range of results in these studies; in Dellavance A. et al.’s work, it is reported that a total of 30,728 serum samples were screened for HEp-2 IFA ANA, and the frequency of anti-DFS70 antibodies was 16.6 [10], while Bizzaro N. et al. indicated that a total of 21,516 serum samples were screened for ANA, and the frequency of anti-DFS70 antibodies was only 0.8 [11]. In this population, a wide range of anti-DFS70 titers is observed, and the frequency of their detection is influenced by factors such as gender (more frequent in women), age, geographical region, and the method of determination [9]; however, these results need to be studied on larger samples to identify clinically significant results. The observed differences in the prevalence of anti-DFS70 antibodies are likely due to differences in analysis methods and the selection of the study population. There have also been mixed results in studies that explored the relationship between the frequency of detecting anti-DFS70 in healthy individuals and age: one study found higher occurrence in individuals under 35 years old among 597 healthy hospital workers [12], other researchers showed that the frequency of anti-DFS70 occurrence is 32% in individuals aged from 18 to 30, which increases to 42% in the 31–40 age group, decreases to 36% in the 41–50 age group, and drops to 10% in those over 50 [13]. At the same time, another study found isolated anti-DFS70 in only 2.1% of healthy children [14]. Prospective studies have also been conducted to monitor the health status and antibody titer dynamics in healthy individuals with confirmed presence of anti-DFS70; for example, a four-year observational study did not register any cases of SARD among 41 healthy individuals with permanent high levels of isolated anti-DFS70 and no other autoantibodies in their blood serum [13]. In a 10-year follow-up study by Gundín S. et al. of 181 patients with positive anti-DFS70, antibody results showed that none of them developed SARD during the observation period [15].
Aleksandrova et al. examined the frequency of detecting anti-DFS70 antibodies in the sera of 45 healthy donors and 12 patients with SLE. Among ANA-positive individuals, 15.6% of healthy volunteers and 100% of SLE patients exhibited positive results. Classical ANA patterns with homogenous, speckled, mixed fluorescence types, and absence of antibodies to anti-DFS70 were observed in 100% of SLE patients and 6.7% of healthy individuals. Monospecific antibodies to anti-DFS70 without classical ANA patterns were detected in 8.9% of healthy individuals and were absent in SLE. Among ANA-positive healthy individuals, the frequency of isolated detection of antibodies to anti-DFS70 was 57%. The authors concluded that monospecific antibodies to anti-DFS70 serve as a negative serological marker for SLE [16]. However, in some studies, an assessment of their potential association with serological and clinical manifestations and disease activity has been conducted. So, in an early SLE (15 months from diagnosis) involving a multinational cohort of patients from 11 countries (n = 1137), anti-DFS70 antibodies were identified in 7.1% of cases, with isolated anti-DFS70 especially in the absence of antibodies against double-stranded DNA and other extractable nuclear antigens found in 1.1% of cases and multivariate analysis showed an association between anti-DFS70 and musculoskeletal manifestations of SLE, and concentration of antibody levels against β2-glycoprotein-1, as well as inverse correlation with anti-dsDNA and anti-La/SSB antibodies [17]. In contrast, Mahler et al. [4] did not find an association between anti-DFS70 and clinical or immunological manifestations of SLE. An analysis of six studies involving 1396 SLE patients showed a frequency of anti-DFS70 antibody occurrence of 2.7% when detecting anti-DFS70 antibodies in the absence of SLE-specific antibodies, and only 0.7% of patients had this combination. Therefore, the exclusive detection of anti-DFS70 antibodies can be considered an exclusion criterion for diagnosing SLE in ANA-positive patients with nonspecific symptoms such as arthralgia, weakness, or rash [7].
The data from a limited number of studies assessing anti-DFS70 antibodies in patients with SSc also indicate a low frequency of detecting this antibody; furthermore, the conclusion is drawn that the concurrent absence of SSc-specific antibodies in individuals with nonspecific symptoms and suspicion of SSc makes this scenario unlikely.
For SjS patients, a low frequency of mono-carriage of anti-DFS70 antibodies has also been established; this finding also allows for its utilization in routine practice as a negative predictor for disease development in this situation. However, a notable feature of SjS is the relatively high frequency of detecting anti-DFS70 antibodies alongside anti-Ro/SS-A antibodies.
For inflammatory myopathies (PM/DM and sporadic inclusion body myositis), the information is extremely limited; in the available studies, DFS70 antibodies were generally diagnosed in a low percentage of cases and were also more commonly associated with patients carrying myositis-specific autoantibodies.
For patients with undifferentiated connective tissue disease, according to the currently available data, a higher frequency of diagnosis of anti-DFS70 antibodies (10.8–12%) is characteristic [7] compared to other SARDs; however, considering the small number of studies, this idea should be examined in more comprehensive research. However, this is complicated by the low frequency of occurrence of this pathology.
Also, when making decisions in clinical practice, it is important to remember that anti-DFS70 can be detected in the blood of patients with conditions other than SARDs. So, elevated levels of anti-DFS70 can be diagnosed in eye diseases (cataracts, atypical retinal degeneration, sympathetic ophthalmia, uveomeningeal syndrome (Vogt–Koyanagi–Harada syndrome), Behcet’s disease, and others); in this situation, a protective role of the DFS70/LEDGF/p75 antigen towards eye structures (lens, retinal pigment epithelial cells) is assumed in response to stress or damage [7]. An association with conditions like interstitial cystitis, bronchial asthma, atopic dermatitis, alopecia areata, chronic fatigue syndrome, prostate cancer, and others has also been identified [4][18][19][20]. However, the determination of anti-DFS70 has not become a routine part of the diagnostic process for these conditions. There is a compelling assumption that this autoantigen may not be a growth factor but rather a protein responding to stress or damage, which is ubiquitously expressed in mammalian cells and tissues, with increased expression in cancer cells and tumors [21].
Therefore, at the present moment, it is recommended to adhere to the following algorithm for utilizing the anti-DFS70 test in the diagnosis of SARD.
  • In the case of a positive test for ANA via the HEp-2 IFA method with a DFS70 fluorescence pattern, an anti-DFS70 test should be conducted;
  • In the case of a positive test for ANA via the HEp-2 IFA method with a fluorescence pattern other than DFS70, it is recommended to conduct an analysis for specific autoantibodies, such as by immunoblotting;
  • In the case of a negative ANA result via the HEp-2 IFA method in conjunction with the absence or presence of anti-DFS70, the likelihood of SARD is minimal;
  • In the case of a positive ANA result via the HEp-2 IFA method in conjunction with the presence of anti-DFS70, the likelihood of SARD is moderate;
  • In the case of a positive ANA result via the HEp-2 IFA method in conjunction with the absence of anti-DFS70, the likelihood of SARD is high.
Therefore, the detection of anti-DFS70 in ANF-positive patients without clinical and/or serological markers characteristic of a specific SARD can be considered a potential marker for excluding the diagnosis of SARD, especially in the early preclinical period. However, the duration of this period, primarily characterized by elevated ANA titers, is not precisely defined and can vary from a few months to several years until the influence of exogenous or endogenous triggering factors leads to the development of clinical symptoms. Detecting both predictors of high-risk development of specific SARD and “excluding” markers at an early stage remains a relevant task for practicing physicians.

This entry is adapted from the peer-reviewed paper 10.3390/biologics3040019

References

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