The role of ANCA testing in ANCA-associated vasculitis

Using patient case studies Dr Eithne Nic an Ríogh and Ms Angel George illustrate the application of ANCA
testing in clinical practice

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a rare multisystem autoimmune disease, characterised by necrotising pauci-immune small vessel vasculitis (SVV). This systemic disease can lead to significant organ damage, primarily affecting the kidneys, lungs, ears, nose and throat, and musculoskeletal systems.¹

AAV comprises three clinico-pathological syndromes: Granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). The estimated overall annual incidence of AAV is 13-20 cases/million, with a prevalence of 300-421 cases per million.²

AAV poses significant clinical challenges due to its multisystem involvement. This article explores the role of ANCA in the diagnosis, treatment and monitoring of AAV.

Identification of ANCA

In 1982, the first case series of patients with antibodies targeting the neutrophil in renal disease was published in Australia.³ Eight patients with necrotising glomerulonephritis were noted to have a factor in their serum that stained the neutrophil cytoplasm. In these cases, routine antibodies were negative.

Soon thereafter the association between ANCA and SVV was defined, marking a significant leap in the understanding and management of this condition.

Vasculitis was initially classified by vessel size in the International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Subsequently, in 2012, a revision incorporated ANCA status into the classification and small vessel vasculitis was divided into AAV and immune complex SVV. This categorisation reflects a greater level of confidence that ANCAs are of pathogenic and diagnostic significance in AAV.

Two main types of ANCAs are relevant in AAV:

1. Cytoplasmic ANCA (c-ANCA): Targets proteinase 3 (PR3), most commonly associated with GPA.

2. Perinuclear ANCA (p-ANCA): Targets myeloperoxidase (MPO), more commonly associated with MPA and EGPA.

Pathogenic role of ANCA

The presence of these antibodies in the serum of patients is a critical factor in both diagnosing and managing AAV, though the exact pathogenic role of ANCA remains an area of active research. Clinical evidence supporting the pathogenicity of ANCA is evident in patients with AAV, where ANCA target antigens (PR3, MPO) have
been localised within or around lesions of fibrinoid necrosis.⁴

The mechanism by which ANCAs cause vasculitis involves ANCA-mediated excessive activation of neutrophils which results in injury to small blood vessels, resulting in an inflammatory cascade involving the release of cytokines, reactive oxygen species and lytic enzymes, and the formation of neutrophil extracellular traps (NETs).^5,6 The strongest evidence for MPO-ANCA pathogenicity is derived from animal models in which immunisation with MPO or transfer of anti-MPO antibodies results in vasculitis.^7,8

Anti-MPO and anti-PR3 subgroups are genetically distinct, suggesting this subclassification is meaningful. Knowledge of the ANCA subtype is more relevant than the phenotypic diagnosis of GPA versus MPA when predicting clinical presentation, disease course, patterns of biomarker regulation, treatment response to rituximab, and comorbidities. Therefore, for research purposes and, increasingly in clinical practice, the stratification of patients with AAV is usually based on ANCA subtype.^5,9

ANCA Testing

ANCA testing is a cornerstone of the diagnostic process for AAV and is useful in differentiating AAV from other vasculitic and inflammatory conditions. While delays in diagnosis are reported by approximately 60 per cent of patients with AAV, more reliable ANCA testing has played a role in improving patient outcomes and survival.^9,10 

For patients with signs and symptoms of AAV, ELISA ANCA antibody testing should be performed as the first step. A positive ANCA test, coupled with clinical features of vasculitis, strongly supports a diagnosis of AAV. ELISA identification of anti-MPO ANCA or anti-PR3 ANCA has higher diagnostic precision than p-ANCA or c-ANCA patterns detected on immunofluorescence.^11,12

The latest consensus guidelines from the European Alliance of Associations for Rheumatology (EULAR) and the organisation Kidney Disease: Improving Global Outcomes (KDIGO) emphasise the importance of initiating treatment immediately in patients with clinical signs of AAV and positive ANCA serology, prior to confirming the diagnosis with a kidney biopsy.^13,14 This approach prevents delays that could lead to rapid disease progression and organ damage. 

However, ANCA testing is not without its limitations. Importantly, ANCA negativity does not exclude the diagnosis of AAV and approximately 10-30 per cent of patients with active GPA (particularly those with upper airway restricted disease) and 5 per cent of patients with active MPA can be ANCA negative.

Additionally, ANCA positivity can be seen in other autoimmune diseases and infections and may also be drug induced, with cocaine being by far the most common culprit.

Anti-glomerular basement membrane (anti-GBM) antibodies should also be tested in patients with pulmonary-renal syndrome as dual positive (anti-GBM/ANCA overlap) cases occur and plasma exchange may be beneficial for these patients.

Estimating relapse risk

Beyond its diagnostic utility, ANCA testing has less clear prognostic implications for long-term management of AAV. Patients with anti-PR3 positive AAV are at higher risk of relapse, although the risk of chronic kidney disease is lower.

Negative switch

Anti-MPO ANCA and anti-PR3 ANCA negativity after treatment is a reassuring signal. Sanders et al studied a cohort of 87 patients with anti-PR3 positive AAV and found that among patients who were persistently negative for anti-PR3 after induction of remission, the risk of relapse is significantly reduced.¹⁶

Moura et al describe how patients with persistently negative MPO-ANCA are at low risk for relapse, even when immunosuppression medications are discontinued.¹⁷

ANCA persistence or reappearance

Relapse risk is higher among those in whom ANCA reappears post induction, although this is insufficiently precise to guide treatment in isolation. Similarly, patients with persistent ANCA positivity are at higher risk of relapse.^18,19 

Trends in ANCA levels

The relationship between serial ANCA levels and disease activity has been the subject of numerous studies and, while not straightforward, patterns have emerged that can inform patient management.

The association between ANCA level and disease activity is strongest in those with vasculitic (eg, glomerulonephritis, mononeuritis) rather than granulomatous dominant inflammation.²⁰ A meta-analysis by Tomasson et al found that a rise in ANCA during remission is only modestly predictive of future disease relapse.²¹ Mukhtyar et al highlighted that while ANCA titres can predict relapses in some patients, they are not universally reliable, emphasising the need for clinical correlation when using ANCA levels to guide treatment.²²

On the other hand, Boomsma et al highlighted that a rise in ANCA levels often precedes clinical relapse by several months, suggesting that regular monitoring could enable pre-emptive treatment and potentially reduce the severity of relapses.²³ Similarly, Han et al found that serial measurements of anti-PR3 and anti-MPO titres in patients with AAV during remission can help predict relapses. They also described how pre-emptive increases in immunosuppression following four-fold titre rises reduced the risk of relapse.

Mehta et al published a valuable systematic review and meta-analysis on the role of ANCA, which supports the use of serial ANCA monitoring as a biomarker for predicting relapse. Overall, the literature suggests that while ANCA testing provides valuable prognostic information, this is not straightforward and reflects the substantial heterogeneity of AAV.

Patients with end stage renal disease should be considered for renal transplantation whether or not ANCA remains positive. Patients who are in remission for less than a year before receiving their allograft have a higher mortality.⁵ Carl can be reassured that after transplantation, the rate of relapse is quite low on current post-transplant immunosuppressive regimens (2.8% per patient-year), however, he should be warned that should a relapse occur, there is potential for graft loss.⁵

Future directions

To conclude, despite its utility, ANCA testing is not without challenges. When considering the role of ANCA in clinical practice we consider that regular testing of ANCA titres is valuable in assessing and treating patients with AAV. While most beneficial in patients with renal involvement, we measure ANCA titres several times per year in patients with AAV. A rise in ANCA titres merits closer follow up, with escalation in immunosuppression if any symptoms develop. Understanding the molecular mechanisms by which ANCA contributes to vasculitis could lead to the development of more specific therapies that target the underlying disease process without broadly suppressing the immune system. As research continues to evolve, the role of ANCA testing will likely become even more refined, offering hope for better outcomes in patients with AAV.

Key Messages:

1. ANCA testing should be performed early in patients with clinical suspicion of AAV.

2. ELISA is the preferred laboratory technique for identifying ANCA. If ELISA is equivocal, follow up immunofluorescence should be performed.

3. Patients with anti-PR3 AAV have higher risk of relapse, while patients with anti-MPO AAV have higher mortality.

4. Trends in ANCA levels can be helpful in guiding treatment course. A four-fold rise in ANCA titres or a negative to positive switch should prompt closer monitoring, potentially including home urine dipstick monitoring.

5. Patients with AAV should be cared for in centres of excellence for AAV. Most importantly, patients should have a reliable point of contact in the hospital to facilitate prompt review should symptoms change.

6. It is beneficial for patients to have access to education focusing on the impact of AAV, its prognosis, key warning symptoms, and treatment. Vasculitis Ireland Awareness provide useful information as well as support for patients and their families. (www.vasculitis-ia.org)

CASE STUDIES

The interpretation of ANCA results is nuanced. To explore this we have outlined four case studies to explore ANCA results through the lens of patient cases. These individual scenarios highlight the varied presentations of AAV and how ANCA results can influence clinical management.

Case 1
Mary is a 65-year-old female diagnosed with anti-MPO-positive AAV. She initially presented with fatigue, weight loss, and significant joint pains. She received IV cyclophosphamide and prednisolone as induction treatment, followed by azathioprine maintenance treatment. While anti-MPO titre was initially positive after two years of treatment, it became negative.

Considering that Mary’s symptoms are well controlled and ANCA is now negative, a trial of immunosuppression would be reasonable. Immunosuppression cessation can be considered two to three years post-diagnosis in a stable patient. When immunosuppression is stopped it is important that Mary is aware of any new symptoms that may arise, which may suggest a relapse. Institution of home dipstick assessment for blood or protein is advised. She should also have contact details for her vasculitis clinic so that she can get in touch with her clinical team promptly if necessary.

Case 2
Robert is a 57-year-old male with anti-PR3-positive AAV with sinonasal and renal involvement. He received rituximab and prednisolone as induction and maintenance treatment, with notable improvement in his symptoms. Anti-PR3 levels remain stable and Robert has no lingering symptoms three years post-diagnosis. Robert is keen to reduce immunosuppression as he is an avid traveller and he worries about picking up a viral illness on flights.

Robert has persistent positive anti-PR3 titre, but no clinical evidence of disease activity. Considering Robert’s clinical picture, it may be worth stopping rituximab, although it is worth remembering that patients who are anti-PR3 positive have a higher risk of relapse. Like Mary, Robert should be well informed on symptoms to be aware of and can consider performing urine dipsticks. A four-fold rise in ANCA titre merits enhanced clinic surveillance and a low threshold for re-introduction of rituximab.

Case 3
Linda is a 62-year-old female with anti-MPO positive AVV, primarily affecting her nerves. She received rituximab and prednisolone as induction treatment. After one year, anti-MPO titre is negative, and rituximab is stopped the following year. Ten months later, the anti-MPO titre becomes positive although Linda remains well.

Considering this negative to positive switch in ANCA titre, Linda is at higher risk of relapse. Having a low threshold to re-introduce immunosuppression is wise in this case and as above, trends in ANCA titres can be helpful. However, immediately recommencing immunosuppression in the absence of clinical symptoms can lead to unnecessary immunosuppression and side-effects, such as infection, that can follow. In this instance, observing Linda’s symptoms closely as well as monitoring trends in ANCA titres is advisable.

Case 4
Carl is a 59-year-old male with anti-MPO-positive AAV. Carl presented two years ago with severe acute anuric kidney injury requiring acute dialysis and treatment with IV cyclophosphamide and plasma exchange. Unfortunately, there was little improvement in Carl’s renal function and he continues to require dialysis, which he performs at home and is keen to receive a renal transplant. He wonders if he is at risk of relapse after transplantation as he has heard that vasculitis can affect the transplanted kidney. Carl remains anti-MPO positive.

References