Autoantibodies against phospholipase A2 receptors (PLA₂R) are a new, highly specific diagnostic marker for primary membranous nephropathy (MN). Detection of anti-PLA₂R using easy-to-perform and inexpensive serological assays can indicate primary MN in patients suffering from nephrotic syndrome and secure a differential diagnosis from secondary MN. Anti-PLA₂R analysis is also useful for determining the disease activity, assessing the extent of treatment required and monitoring responses to therapy. Anti-PLA₂R antibodies can be determined using innovative indirect immunofluorescence and ELISA test systems.

by Jacqueline Gosink, PhD

Primary membranous nephropathy
Primary MN, also known as primary membranous glomerulonephritis or primary MGN, is a chronic inflammatory autoimmune disease of the blood-filtering structures of the kidneys (glomeruli). It is accompanied by a progressive reduction in renal function. The disease manifests the complex nephrotic syndrome, which is characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia, edema and lipiduria. Primary MN is one of the leading causes of nephrotic syndrome in adults. As proteinuria increases, so does the long-term risk of kidney failure with major morbidity and mortality, especially from thromboembolic and cardiovascular complications. Around a third of patients progress to end-stage renal disease, a third exhibit persistent proteinuria without progression to renal failure, and the remainder experience spontaneous remission. Primary MN is prevalent in all ethnic groups and in both genders, with men over 40 years old being the most frequently affected.

Diagnostic challenge
The diagnosis of primary MN is demanding, as the disease must be differentiated from other nephropathies, especially from secondary MN, which is triggered by an underlying cause such as a malignant tumour, an infection, drug intoxication or another autoimmune disease such as systemic lupus erythematosus or diabetes mellitus type 1. Of all MN cases, 20-30% are of secondary genesis, while the remaining 70–80% are classified as primary. Primary cases with no detectable anti-PLA₂R antibodies are subclassified as idiopathic; it has been postulated that these patients may exhibit antibodies against other, as yet unidentified, target antigens. Reliable differentiation of primary and secondary forms of MN is critical because of different treatment regimes: primary MN is treated with immunosuppressants, while therapy for secondary MN is targeted at the underlying disease.

MN is diagnosed by kidney puncture followed by histological examination or electron microscopy of the tissue to identify the characteristic glomerular immune deposits. To obtain a definite diagnosis of primary MN, secondary causes must be excluded, which involves additional time-consuming and often invasive procedures, for example tumour screening. Moreover, in some patients, MN appears before the secondary cause is even detectable, adding an extra layer of complexity to diagnosis and therapeutic decision-making. Primary MN must also be differentiated from other autoimmune diseases with kidney involvement, for example lupus nephritis, vasculitides associated with antibodies against neutrophil cytoplasm (ANCA) and Goodpasture’s syndrome. The availability of reliable serological tests to support the diagnosis of primary MN has been elusive until recently due to lack of knowledge about the target antigen.

New pathognomonic marker
Autoantibodies against PLA₂R were first discovered and described in patients with primary MN in 2009 (1). PLA₂R is a transmembrane glycoprotein (Figure 1) which is expressed in human glomeruli on the surface of podocytes and is involved in regulatory processes in the cell following phospholipase binding (Figure 2). Type M PLA₂R has been identified as the major target antigen of autoantibodies. In patients with primary MN, antigen-antibody complexes form deposits in the glomerular basement membrane, where they trigger local complement activation with overproduction of collagen IV and laminin. This causes damage to the podocytes, via destruction of the cytoskeleton and broadening of the basement membrane. As a result protein enters the primary urine, giving rise to proteinuria and other symptoms.

Differential diagnosis
Autoantibodies of class IgG against PLA₂R are present in the serum of up to 70-80% of patients with primary MN (1, 2), whereas they are not found in healthy blood donors or patients with secondary MN or other kidney diseases such as lupus nephritis (3) or IgA nephritis. The high predictive value of anti-PLA₂R makes this parameter ideally suited as a diagnostic marker (4).

Disease evaluation
Anti-PLA₂R antibodies are, moreover, very sensitive markers of clinical disease activity. They reflect the pathogenic immunological activity of the disease, which is responsible for the clinical expression in the form of proteinuria. High antibody titres indicate a severe disease course (2, 5), while low titres are associated with a decreased risk of renal failure and a greater rate of spontaneous remission (6). Thus, the anti-PLA₂R titre also serves as a prognostic indicator.

Therapy monitoring
Treatment with immunosuppressants results in a drop in the anti-PLA₂R titre, while in relapse the antibody titre increases again. Significantly, changes in the antibody titre typically precede changes in the proteinuria (6, 7). Thus, a titre increase is detectable before proteinuria appears, while a titre decrease is observed before a reduction in the proteinuria. Patients in remission exhibit residual proteinuria months after the anti-PLA2R titre becomes undetectable. Anti-PLA₂R measurements are therefore extremely useful for early therapeutic decision-making and for long-term monitoring of responses to immunotherapy.

A recently published prospective study reinforced the value of anti-PLA₂R antibodies as a marker of clinical outcome (8). In the study 133 patients with primary MN were tracked over a time period of 24 months. In all cases there was a clear correlation between proteinuria and anti-PLA₂R levels. In patients who were given immunotherapy, a significant time lag was observed between the rapid fall in antibody levels and the protracted reduction in proteinuria. Moreover, remission of proteinuria occurred later in individuals with high antibody levels than in those with low levels. In patients who did not receive immunosuppressive therapy, spontaneous remission was also associated with a reduction in anti-PLA₂R, while individuals who did not achieve remission showed continued elevated antibody levels. Thus, anti-PLA₂R proved a reliable biomarker for immunological and clinical activity in primary MN.

Risk assessment
Up to 40% of patients with primary MN experience a relapse after kidney transplantation. The risk of recurrent primary MN is particularly high if anti-PLA₂R antibodies are found prior to transplantation. In a study on a patient with primary MN, who exhibited high anti-PLA₂R levels before and three months after transplantation (7), it could be shown that immunotherapy resulted in a drop in the antibody concentration and also the level of proteinuria. Other studies have shown that anti-PLA₂R or PLA₂R deposits are detected more often in transplant patients with recurrent MN than in those with de novo MN. A retrospective analysis of fifteen transplant patients revealed that a persistently positive anti-PLA₂R activity at six months or later after transplantation was a significant risk factor for relapse, especially in patients on a weak immunosuppressive regimen (9). Thus, the anti-PLA₂R antibody titre is useful for assessing the risk of relapse after transplantation and the extent of immunotherapy needed to prevent a recurrence.

Anti-PLA₂R test systems
Anti-PLA₂R autoantibodies can be determined easily and reliably using standardized indirect immunofluorescence test (IIFT) and ELISA systems. In the IIFT a BIOCHIP of transfected human cells expressing recombinant PLA₂R is used as the antigenic substrate to provide monospecific antibody detection (Figure 3). A second BIOCHIP containing cells transfected with an empty vector serves as a control. The IIFT represents an established test for serodiagnostic screening, providing qualitative and semi-quantitative antibody analysis. The corresponding ELISA is based on purified recombinant PLA₂R and shows the same high-quality characteristics as the IIFT. The ELISA is particularly useful for disease and therapy monitoring as it offers precise quantification of antibody levels in patient sera. The IIFT and ELISA are fast and simple to perform and are suitable for use in any diagnostic laboratory. Both procedures can be automated.

Clinical data
The performance characteristics of the Anti-PLA₂R IIFT and ELISA have been assessed in a multitude of studies. In a retrospective clinical study (10) the Anti-PLA₂R IIFT yielded a prevalence of 52% in a cohort of 100 patients with biopsy-proven primary MN, and a specificity of 100% with respect to healthy controls and patients with secondary MN or non-membranous glomerular injury. In a prospective clinical study (11) the sensitivity amounted to 82% in patients with biopsy-proven MN where no secondary cause could be found. The difference in sensitivities obtained in different study panels is most likely due to factors such as disease remission and the therapy status of the individuals, which can influence the antibody results, especially when studies are performed retrospectively.

Results obtained with the ELISA show a very good correlation with results from the IIFT (Figure 4). In a retrospective study with sera from 198 patients with primary MN and 836 healthy and disease controls, the ELISA showed a sensitivity of 96% with respect to the IIFT, and a specificity of 99.9% with borderline sera included (12). The few discrepant sera that were negative in the ELISA gave only low titres of 1:10 to 1:100 in the IIFT. All sera with titres of over 1:100 in IIFT were also positive in ELISA. 

Summary
Antibodies against PLA₂R represent a landmark development in nephrological diagnostics. Their detection can secure a diagnosis of primary MN in patients with nephrotic syndrome, offering a convenient, non-invasive alternative to biopsy. Anti- PLA₂R determination, moreover, yields information about the disease status and evolvement. Serial measurements are especially useful for monitoring therapy responses over the long term and guiding decision-making on the extent of treatment required for individual patients. Serological tests based on state-of-the-art IIFT and ELISA technology provide simple, quick and highly specific anti-PLA₂R antibody detection.

References
1. Beck et al. N. Engl. J. Med. 2009: 361: 11-21
2. Hofstra et al. Clin. J. Am. Soc. Nephrol. 2011: 6: 1286-91
3. Gunnarsson et al. Am. J. Kid. Dis. 2012: 59 (4): 585-6
4. Schlumberger et al. Autoimmunity Reviews 2014: 13: 108-13
5. Kanigicherla et al. Kidney Int. 2013: 83: 940-948
6. Hofstra et al. J. Am. Soc. Nephrol. 2012: 23 (10): 1735-43
7. Stahl et al. N. Engl. J. Med. 2010: 363: 496-8
8. Hoxha et al. J. Am. Soc. Nephrol. 2014: 25: 1137-9
9. Seitz-Polski et al. Nephrol. Dial. Transplant. 2014: under revision
10. Hoxha et al. Nephrol. Dial. Transplant. 2011: 26 (8): 2526-32
11. Hoxha et al. Kidney Int. 2012: 82: 797-804
12. Daehnrich et al. Clin. Chem. Acta. 2013: 421C: 213-8

The author
Jacqueline Gosink PhD
EUROIMMUN AG
Seekamp 31
23560 Luebeck
Germany

E-Mail: j.gosink@euroimmun.de

B-cell maturation antigen (BCMA) was originally identified as a cell surface receptor expressed on late-stage B cells, plasma cells, and B-cell malignancies including multiple myeloma (MM). We recently discovered that BCMA is shed into the blood of MM patients, and, therefore, serum BCMA may serve as a new prognostic marker to track disease status and response to treatment.

by Eric Sanchez, Suzie Vardanyan, Mingjie Li, Cathy Wang, Dr Haiming Chen, and Dr James R. Berenson

Background
B-cell maturation protein (BCMA, also referred to as TNFRSF17 or CD269) is a receptor shown to be expressed in B lymphocytes and at increasing levels as these cells mature [1]. BCMA binds the ligands BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand) [2, 3]. Membrane-bound expression of BCMA has been demonstrated in human CD138-expressing cells from multiple myeloma (MM) bone marrow (BM) and MM cell lines [4]. It has also been shown to be expressed on malignant cells from Hodgkin’s lymphoma and Waldenstrom’s macroglobulinemia (WM) patients [5, 6]. Although the response to treatment of MM patients is traditionally followed with measurement of their monoclonal immunoglobulin (Ig) levels, some patients do not produce this marker; and, moreover, in others, it is lost during the course of their disease.

Serum BCMA levels are elevated in MM patients
We measured serum BCMA by ELISA from patients with newly diagnosed MM, monoclonal gammopathy of undetermined significance (MGUS) and healthy control subjects. Using the International Staging System, 30, 12 and 7 patients with stages 1, 2 and 3, respectively, and one with unknown staging were analysed. We found that the serum levels of BCMA in MM patients (13.87 ng/ml) were elevated when compared to healthy controls (2.57 ng/ml; P <0.0001) and MGUS individuals (5.30 ng/ml; P = 0.0157; Fig.1A). We then determined that serum BCMA levels correlated with the MM patient’s response to therapy. Patients responding to their treatment regime had lower serum BCMA levels than patients with progressive disease (P = 0.0038; Fig. 1B). To confirm that the BCMA found in the blood of MM patients came from cells within the BM, BM aspirates were obtained from MM patients and BM mononuclear cells (MCs) were isolated and cultured for 48 hours. MM patients showed high levels of BCMA in culture medium whereas healthy subjects lacked significant amounts of BCMA.  Moreover, serum and supernatant BCMA levels from MM patients were compared and showed a strong correlation (r = 0.82) between the levels in the serum and supernatants from cultured MM BMMCs (Fig. 2). To exclude the possibility that the BCMA detected in MM patient serum and from cultured MM BMMCs may have been derived from non-malignant cells, we evaluated human BCMA levels in our human MM xenografts growing in severe combined immunodeficient (SCID) mice.  Animals were implanted with the human MM tumour LAGκ-2 and analysed for human serum BCMA levels. SCID mice dosed with bortezomib (0.5 mg/kg, twice weekly) had a reduction in tumour volume compared to untreated mice (P = 0.0067; Fig. 3A), and human serum BCMA levels from these bortezomib-treated animals were also markedly lower compared to untreated mice (P = 0.0006; Fig. 3B). Similar results were obtained when using our other MM xenograft models (LAGλ-1, LAGκ-1A). Human BCMA was not detected in the serum of non-tumour-bearing mice (data not shown). A rise in serum BCMA levels from the possible release of membrane-bound protein from dead MM cells was not observed in mice following drug treatment. Thus, it can be concluded that the serum levels of BCMA in the mice are derived from live MM cells. Soluble BCMA has been shown to inhibit normal B-cell development through interference with the binding of its ligands (BAFF and/or APRIL) to membrane-bound BCMA on normal B cells. One group demonstrated that administration of BCMA–Ig fusion protein to normal mice, which inhibits the binding of BAFF to B cells, resulted in a dramatic reduction in B-cell numbers in the blood and peripheral lymphoid organs [2]. Splenic B-cell reductions were shown to occur in an in vitro mouse splenocyte proliferation assay following in vivo administration of BCMA–Fc fusion protein to normal mice [7]. Other investigators have shown that injecting soluble BCMA–Fc fusion protein, which binds BAFF and APRIL both as free and membrane-bound ligands, into nude mice bearing human colon or lung carcinoma cell lines resulted in inhibition of tumour growth [3]. The authors suggested that BCMA bound its ligand APRIL, and prevented the known stimulatory effect of APRIL on these cell lines. Additionally, investigators have shown in vitro the existence of BCMA–BAFF complexes [3, 7, 8]. In the context of cancer growth, one group demonstrated that BCMA injected into mice reduced the growth of tumour cells from human colon or lung carcinoma cell lines in vivo by binding its ligand APRIL [3]. Thus, we are currently conducting studies to determine if such complexes exist in vivo and may block the immune function of myeloma patients.

Conclusions and future directions
Measurement of MM tumour mass is indirect, given the location of this BM based malignancy. Thus, assessment of tumour burden in response to therapy is difficult but essential to effectively monitor patients with MM. Traditionally, changes in Ig levels have been used to follow disease progression and response to treatment. In fact, for three decades Ig was a prognostic factor used in the Durie-Salmon staging system [9], which, until recently, was the most widely used staging system [10].  However, assessment of this protein does not always accurately reflect changes in MM tumour burden [11–13]. Additionally, small subsets of MM patients do not produce this marker. Thus, additional markers are needed to assess response to therapy in these non-secretory patients and in MM patients as a whole.

We have now shown that BCMA is present in the serum of MM patients; and, moreover, its levels correlate with the patient’s response to therapy [14]. We have also previously shown that supernatant from cultured BMMCs from MM patients with active disease contain much higher levels of this protein in their culture medium than in healthy subjects and those with MGUS or indolent MM [14]. SCID mice bearing human MM xenografts also showed high levels of BCMA in their sera, and these levels decreased in response to anti-MM therapy. We believe that that serum BCMA will eventually be used in the clinic as a diagnostic and prognostic marker for MM patients.

References
1. Laabi Y, Gras MP, Brouet JC, et al. The BCMA gene, preferentially expressed during lymphoid maturation, is bidirectionally transcribed. Nucleic Acids Res. 1994; 22(7): 1147–1154.
2. Thompson JS, Schneider P, Kalled SL, et al. BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population. J Exp Med. 2000; 192(1): 129–135.
3. Rennert P, Schneider P, Cachero TG, et al. A soluble form of B cell maturation antigen, a receptor for the tumor necrosis family member APRIL, inhibits tumor cell growth. J Exp Med. 2000; 192(11): 1677–1684.
4. Novak AJ, Darce JR, Arendt BK, et al. Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival. Blood 2004; 103(2): 689–694.
5. Chiu A, Xu W, He B, et al. Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL. Blood 2007; 109(2): 729–739.
6. Elsawa SF, Novak AJ, Grote DM, et al. B-lymphocyte stimulator (BLyS) stimulates immunoglobulin production and malignant B-cell growth in Waldenstrom macroglobulinemia. Blood 2006; 107(7): 2882–2888.
7. Pelletier M, Thompson JS, Qian F, et al. Comparisons of soluble decoy IgG fusion proteins of BAFF-R and BCMA as Antagonist for BAFF. J Biol Chem. 2003; 278(35): 33127–33133.
8. Shu HB, Johnson H. B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1. Proc Natl Acad Sci U S A 2000; 97(16): 9156–9161.
9. Durie BG, Salmon SE. A clinical staging system for multiple myeloma: correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer 1975; 36(3): 842–854.
10. Larson RS, Sukpanichnant S, Greer JP, et al. The spectrum of multiple myeloma: diagnostic and biological implications. Hum Pathol. 1997; 28(12): 1336–1347.
11. Sullivan PW, Salmon SE. Kinetics of tumor growth and regression in IgG multiple myeloma. J Clin Invest. 1972; 51(7): 1697–1708.
12. Kawano M, Huang N, Harada H, et al. Identification of immature and mature cells in the bone marrow of human myelomas. Blood 1993; 82(2): 564–570.
13. Zaanen HCT, Lokhorst HM, Aarden LA, et al. Chimaeric anti-interleukin 6 monoclonal antibodies in the treatment of advanced multiple myeloma: a phase I dose-escalating study. Br J Haematol. 1998; 102(3): 783–790.
14. Sanchez E, Li M, Kitto A, et al. Serum B-cell maturation is elevated in multiple myeloma and correlates with disease status and survival. Br J Haematol. 2012; 158(6): 727–738.

The authors
Eric Sanchez BA; Suzie Vardanyan BS; Mingjie Li BS; Cathy Wang BS; Haiming Chen MD, PhD; and James R. Berenson* MD
Institute for Myeloma & Bone Cancer Research, West Hollywood, CA, USA

*Corresponding author
E-mail: Jberenson@imbcr.org