Enhancing Systemic Lupus Erythematosus Diagnosis: Advances in Diagnostic Tools

Systemic lupus erythematosus ( SLE) is a heterogeneous autoimmune disease with a variable clinical course and prognosis. Its signs and symptoms can be subtle or marked, affect a single organ system or several, and change over time, making it a difficult disease to diagnose.

Typical manifestations include skin rashes, including malar "butterfly-wing" rash, arthritis, pleurisy and serositis, alopecia, and lupus nephritis.

Frustratingly for both doctors and patients, response to treatment can be variable and difficult to predict. This clinical heterogeneity probably derives from a complex immunological dysregulation that results in the pathogenesis of the disease.

At the cellular level, this process is driven by interactions between the adaptive and innate immune systems leading to cytokine upregulation, complement activation, immune complex deposition, and ultimately inflammation and tissue damage.

Early diagnosis of SLE is crucial to prevent flares and resulting tissue damage.

Importantly, the path to SLE begins before the disease manifests clinically. Autoantibodies have been found in the serum of patients with SLE approximately 3 to 9 years before diagnosis.

Antinuclear antibodies (ANA), anti-Ro, antiLa, and antiphospholipid antibodies are the first to appear in the serum, and the accumulation of autoantibodies usually stops after the onset of the disease.

Nowadays, ANA tests are widely available, which has improved the diagnostic delay, but the delay is still considerable. A study from the United Kingdom found that 5 years before diagnosis, patients with SLE visited their primary care provider twice as often as patients without SLE, for symptoms such as arthritis, rash, fatigue, serositis, fever, and others.

Delays in diagnosis may contribute to racial disparities in disease outcome, as black and Hispanic patients frequently have more severe manifestations at the time of diagnosis.

ANA are a group of autoantibodies that bind to various nuclear and cytoplasmic antigens.

They are sensitive biomarkers for the evaluation of suspected rheumatic diseases associated with ANA, among which SLE is most common, and the detection of ANA is usually a requirement to participate in clinical trials. It is not useful for monitoring disease activity. There are 3 assays for ANA testing: enzyme immunoassay, multiplex immunoassay, and indirect immunofluorescence assay in HEp-2 cells, the latter being the gold standard. Up to 25% of healthy patients can be ANA positive, which limits the specificity of the screening test.

Most ANA-positive patients never develop rheumatic disease. ANA positivity is more common among women and certain racial and ethnic groups, including African Americans. Many healthy ANA-positive people have antibodies directed at the dense end of speckle antigen 70 (DFS70). These antibodies are extremely rare in patients with suspected ANA-associated rheumatic diseases.

The extractable nuclear antigen panel analyzes specific autoantibodies that react with components of the cell nucleus, revealing 2 to 11 different autoantibodies that aid diagnosis and have prognostic implications.

Apart from the extractable nuclear antigen test, anti-dsDNA (double-stranded DNA) is highly specific for SLE; Antibody levels correlate with disease activity, particularly lupus nephritis. The European Autoimmunity Standardization Initiative has standardized the morphological characteristics of several patterns that correlate between the specific antigen and the disease.

Before 2019, there were 2 main classification criteria for SLE: the 1997 American College of Rheumatology (ACR) criteria, and the Systemic Lupus International Collaborating Clinics Criteria (SLICC). To maintain the specificity of the ACR 1997 criteria while increasing the sensitivity of the SLICC criteria, in 2019 the Alliance of Associations for Rheumatology (EULAR)/ACR) 2019 developed SLE classification criteria for research purposes.

ANA ≥1:80 had a sensitivity rate of 98% for the diagnosis of SLE and was added to the criteria as an entry requirement. Differential weighting of criteria was used in a point system with 10 points indicating SLE classification. On the other hand, it is noted that the criteria only count for SLE if there is no other explanation. The 2019 criteria have been validated in adults and children, with sensitivities of 92% and 89%, respectively.

> Cell-bound complement tests

The newly developed multi-analyte assay panel, commercially called the AVISE test (Exogen Diagnostics), performs a 2-tier test that uses cell-bound complement activation products (CB-CAPs) as biomarkers for diagnosis and disease activity. The test measures C4d autoantibodies bound to erythrocytes and C4d bound to B cells, to aid in the diagnosis of SLE. CB-CAP has greater sensitivity than standard complement and anti-dsDNA measurements alone for SLE in adults and children and predicts probable progression from SLE to SLE classifiable by ACR criteria.

Furthermore, CB-CAP abnormalities may predict a higher SLE severity index in patients with otherwise normal complement. A recent study of 161 patients found that CB-CAP testing increased physician confidence in the diagnosis of SLE and increased the frequency of early treatment with hydroxychloroquine (HCQ).

> Interferon tests

Type I and II interferons are upregulated before classifiable SLE develops, although data to support these findings are limited by small study sizes. It will not be long before functional assays for blood analysis are commercially available. Interferon testing remains valuable research but has not yet proven to be viable as a biomarker in clinical practice.

Standard treatment of SLE uses antimalarial therapy, usually hydroxychloroquine (HCQ), unless there is a contraindication to this medication.

Antimalarials reduce the antigenic load in the lysosome and inhibit the activation of interferon by nucleic acids. HCQ is generally well tolerated and has been shown to reduce the risk of disease flares, improve life expectancy, decrease the risk of thrombosis, and have positive effects on skin diseases and musculoskeletal manifestations of SLE.

It is important to highlight that its early use can be beneficial since in patients with SLE it can reverse the inflammatory changes of cytokines and interferons. During pregnancy of anti-Ro-positive mothers, HCQ reduces the risk of preterm birth and fetal heart block. There are data to support the use of other antimalarial drugs such as chloroquine and quinacrine in SLE, but chloroquine is associated with a higher rate of retinal toxicity while quinacrine is difficult to access, limiting its widespread use. . Side effects of HCQ are gastrointestinal upset and, more rarely, retinal toxicity and cardiomyopathy.

Retinal toxicity can be attenuated by appropriate dosing (5 mg/kg/day) and annual screening after the first 5 years of treatment, using advanced techniques such as optical coherence tomography. Monitoring HCQ blood levels may also be useful in identifying patients at increased risk of retinal toxicity.

Glucocorticoids are traditionally used to rapidly control disease activity. The dosage depends on its severity. In general, 5-10 mg of prednisone equivalents are sufficient for mild manifestations. For more severe cases, higher doses (up to 0.5–1 mg/kg prednisone equivalent, with or without initial pulse of intravenous (IV) methylprednisolone) may be required for lupus nephritis, severe hematologic involvement, or central nervous system disease.

It is recommended to limit steroid use by dosing only what is essential and tapering whenever possible, as they correlate closely with the accumulation of damage over time. In addition to antimalarials, the choice of additional treatments depends on the disease.

There are no therapies specifically approved by the US FDA for cutaneous SLE, so management of the skin disease is based on expert opinion. Other medications may be used such as topical steroids and calcineurin inhibitors, dapsone, methotrexate, lenalidomide, or mycophenolate mofetil (MMF). For arthritis, methotrexate, leflunomide, and MMF may offer benefit and allow steroid dosage reduction. Methotrexate has also been shown to generally improve overall disease activity. Azathioprine is also often used to reduce overall disease activity.

Cyclophosphamide is generally reserved for organ-threatening manifestations such as central nervous system or lupus nephritis. Before the latest advances, lupus nephritis therapy had remained unchanged for a decade. The main drug was cyclophosphamide, used orally in the 1970s and then by IV pulses (0.5–1.0 g/m2) in the 1980s.

A second protocol, called Euro-lupus , applies 6 IV pulses of 500 mg. It has been shown that doses of cyclophosphamide 2 weeks apart were equally effective in achieving remission of renal pathology compared to higher doses of pulse cyclophosphamide.

It is currently the preferred initial option for most patients. After the studies were criticized for only providing data from European (predominantly Caucasian) patients, subsequent trials using Euro-lupus dosing also showed equal response rates in black patients.

A study completed in 2009 demonstrated that a target dose of 3 g/day of mycophenolate mofetil (MMF) and IV pulses of cyclophosphamide achieved the same efficacy in terms of renal response rate, with no differences in adverse events. Thus, MMF has become a standard option for the treatment of lupus nephritis.

While monotherapy with mycophenolate mofetil (MMF) or cyclophosphamide is still considered the standard of care, the FDA has recently approved both dual and targeted therapy.

Belimumab combined with MMF or cyclophosphamide may increase the chance of a partial or complete renal response compared with MMF or cyclophosphamide alone, and is now approved by the FDA for this indication. On the other hand, a calcineurin inhibitor combined with MMF may provide better renal response rates. This has been demonstrated with tacrolimus and voclosporin, which in 2021 also received the FDA label for lupus nephritis.

Tacrolimus may also provide benefit in lupus nephritis as monotherapy, but more studies need to be completed before it can be implemented as standard practice.

> Belimumab . It is a fully human, recombinant monoclonal antibody (mAb) that blocks the binding of soluble B cell stimulator to its receptor on B cells, decreasing the survival, differentiation and activation of those cells. It was the first biologic medication approved by the FDA for SLE and is available for IV infusion or subcutaneous injection.

Four major double-blind phase III randomized controlled trials demonstrated the efficacy of belimumab in patients with active disease on standard treatment. Improvements included response in composite indices, reduction of flares and steroid dosage. A recent review also showed its effectiveness without detecting major harm in the treated patients. A 6-year follow-up study showed that subjects receiving belimumab for SLE saw significant long-term improvements in fatigue and health-related quality of life outcomes. The authors note that patients with organ-threatening disease were excluded from the trials.

More recently, another phase III study evaluated belimumab for the treatment of lupus nephritis. Other studies showed a better primary efficacy renal response and a complete renal response at week 104 in patients treated with belimumab, compared to placebo, and a lower risk of renal events or death in the belimumab group.

> Rituximab . It is a chimeric mAb targeting CD20, a transmembrane protein on all B cells except pro-B cells and plasma cells, resulting in cytotoxicity and B cell exhaustion. Its efficacy in SLE has been demonstrated by the improvement of patients (including lupus nephritis) in several case series and retrospective studies. The efficacy of rituximab was studied in patients with SLE without moderate to severe kidney injury who were on standard treatment, but the study did not meet its primary or secondary endpoints. Subgroup analysis showed higher rates of clinical responses and partial responses among African American and Hispanic patients.

Subsequently, the phase III LUNAR study aimed to study the drug in patients with SLE and class III or IV lupus nephritis. Although the study did not meet primary or secondary endpoints, there were more partial responders in the rituximab group than in the placebo group (31% vs. 15%); no patients required rescue therapy with cyclophosphamide in the rituximab group (compared with 8 patients in the placebo group). Despite the effectiveness of rituximab not being proven in the aforementioned study, doctors still continue to use it, particularly in refractory patients or those suffering from SLE with hematological manifestations, often with excellent results.

> Anifolumab . It is a human mAb targeting subunit 1 of the type I interferon receptor that inhibits signaling of all types of interferons. It is administered by IV infusion and in 2021 it was approved by the FDA for the treatment of SLE. A phase II randomized controlled trial found that anifrolumab reduced disease activity in patients with moderate to severe SLE. However, the first phase III study, TULIP-1, missed the primary endpoint, the LES-4 response rate.

Several secondary endpoints showed response with favorable results. Later, the more comprehensive TULIP-2 study showed a significant reduction in disease activity in patients with moderate to severe SLE. Pooled data for TULIP-1 and TULIP-2 showed a reduction in flares, including those resulting from steroid tapering. Anifrolumab will likely be a useful tool for the treatment of SLE in patients with moderate to severe disease activity, especially cutaneous, who do not tolerate or do not respond to conventional therapies. However, data on real-world effectiveness is still lacking.

> Voclosporin . It is an oral calcineurin inhibitor. It corresponds to the same class of drugs as tacrolimus and cyclophosphamide. It was approved by the FDA in January 2021 for the treatment of active lupus nephritis, combined with immunosuppressive agents. Two pivotal studies have demonstrated better renal response rate and reduction in proteinuria when combined with MMF and steroids, compared to MMF and steroids alone. There are preliminary interim data from a 2-year study that have demonstrated sustained reductions in proteinuria and no change in renal function after up to 30 months of drug exposure.

> Emerging therapies . New interventions are currently being investigated to benefit patients with SLE. Inhibition of several immune-related kinases, including JAK1 and TYK2, has shown promise. Blocking specific cells, such as plasmacytoid dendritic cells, also promises benefits. Strategies to increase regulatory T cells, using low doses of IL-2 and IL-2-like molecules, are being investigated. There is great hope that the next 10 to 20 years of research will be transformative for the management of SLE, as new pathological pathways will have been discovered and new drugs developed.

> Vitamin D supplementation

Vitamin D deficiency and insufficiency are common in patients with SLE and are associated with lack of sun exposure.

Vitamin D deficiency is correlated with increased disease activity, higher levels of fatigue, and increased risk of thrombosis.

In lupus nephritis, vitamin D supplementation can reduce proteinuria and delay the progression of kidney damage. The recommended target level of 25(OH) vitamin D is 40 ng/ml , as higher levels did not show therapeutic benefits. Vitamin D supplementation is well tolerated and levels should be routinely tested to ensure absorption.

> Dietary modifications

In SLE, gut microbiome dysbiosis likely plays a role in disease generation and activity, but needs further investigation. Several studies that made comparisons between different human populations with SLE and healthy people found a lower proportion of Firmicutes and Bacteroidetes. V arious studies in lupus-prone mice have shown that dysbiosis or particular deviation of commensal organisms worsens autoimmune manifestations. In one study, antibiotic-induced changes in the gut microbiota resulted in decreased systemic autoimmunity and improved renal pathology in the murine lupus model.

Despite this evidence of dysbiosis in SLE, diet-microbiome interactions require further study to justify evidence-based recommendations on factors such as probiotics and diet. Furthermore, while there is no agreed upon "lupus diet," various dietary modifications may have beneficial effects. In a cross-sectional study, a Mediterranean diet decreased disease severity and cardiovascular risk. It has also been observed that a diet with higher omega-3 fatty acid intake and a lower omega-6:omega-3 ratio was favorably associated with patient-reported outcomes in both disease and sleep quality.

Exposure to ultraviolet light can induce flares of systemic and cutaneous SLE.

Although the exact mechanisms of UV light-induced autoimmunity remain poorly understood, evidence suggests: generation of reactive oxygen species, increased DNA damage, increased antigen exposure, production of inflammatory mediators including type I interferons and, greater recruitment of inflammatory cells.

The use of broad-spectrum sunscreens against UV exposure, sun protection factor (SPF) ≥30, is strongly recommended. Other photoprotection methods include seeking shade, avoiding the sun, wearing hats, sunglasses, long sleeves and pants. It is very important to educate the patient about photoprotection.

Glucocorticoids have rapid action to suppress the immune system in SLE flares, but cause toxicity. The goal of administering glucocorticoids is to reduce the dose to ≤7.5 mg/day as quickly as possible and maintain the lowest dose needed.

Short-term complications of glucocorticoids include obesity, hypertension, type 2 diabetes , susceptibility to infections, and irreversible damage, including avascular necrosis and stroke. Long-term consequences include cataracts, osteoporosis, fractures, and cardiovascular disease.

Damage accumulation depends on time and dose. A 15-year SLE cohort study found that 80% of organ damage was possibly or definitely related to glucocorticoid exposure. Recent trials have suggested that limiting cumulative glucocorticoid exposure may not adversely affect outcomes.

In a pilot study, 50 patients with active lupus nephritis received rituximab and MMF, 2 doses of methylprednisolone 500 mg IV, without oral steroids. After 12 months, 53% achieved complete remission, comparable to the results of previous studies with conventional oral steroid use.

A recent phase III study of voclosporin used starting doses of prednisone, 25 mg/day, suggesting that efficacy is not impaired by lower-dose steroid regimens. Additional randomized trials are needed to determine whether low-dose steroid regimens are as effective as conventional therapy.

Cardiovascular diseases and infections account for the majority of mortality associated with SLE.

Cardiovascular risk factors , including hypertension and type 2 diabetes, are more common in patients with SLE, while resistant hypertension is almost twice as common in patients with SLE compared to controls.

Health maintenance screening and prevention of SLE-related complications are essential to providing quality care. Management includes keeping vaccination status up to date; perform age-appropriate routine malignancy screening; hypertension, diabetes and, detection and management of hyperlipidemia; in addition to education regarding self-care and healthy lifestyle strategies.