Anti-PLA2R assay guidance

In 2009, anti-PLA2R antibodies were first described to be a diagnostic marker for Idiopathic Membranous Nephropathy (IMN) (Beck et al, 2009). In 2011, Genome Wide Association Study (GWAS) confirms that the genetic susceptibility to IMN involves PLA2R and HLA DQA1 (Stanescu et al, 2011). In 2013, the first ELISA for assaying anti-PLA2R autoantibodies was reported (Kanigicherla et al, 2013). The major epitope on PLA2R recognised by anti-PLA2R autoantibodies is identified in 2014 (Fresquet et al, 2014).

Clinical research studies support the following statements:

Anti-PLA2R as a diagnostic autoantibody in IMN

  • Phospholipase A2Receptor 1 (PLA2R) is an autoantigen found on podocytes
  • Up to 75% of biopsy proven IMN patients naïve to immunosuppression are seropositive for anti-PLA2R
  • 5-30% of biopsy proven MN with secondary causes (tumour, SLE) naïve to immunosuppression are seropositive for anti-PLA2R

Anti-PLA2R for monitoring disease activity and therapeutic response

  • Anti-PLA2R is reported to be high in active disease and low/normal in complete remission
  • Anti-PLA2R levels respond to immunosuppression often showing an immunological remission (anti-PLA2R in normal range) 3-6 months ahead of remission of proteinuria where this occurs.
  • In patients treated with immunosuppression, if the anti-PLA2R level remains positive at the end of treatment, the patient is at greater risk of relapse
  • Anti-PLA2R levels tend to rise in association with relapse
  • Anti-PLA2R levels have been reported to be associated with recurrent disease post transplant

 Anti-PLA2R and pathogenicity

  • Currently there is no direct evidence that anti-PLA2R cause proteinuria (see later in Q&A)

Assays for anti-PLA2R

  • For clinical assays, ELISA is preferred. Other assays formats have been described e.g. western blotting,  immunofluorescence and bead based assays (ALBIA)

Accreditation of assays

Currently there is no calibrated international standard for anti-PLA2R against which assays can be calibrated and no quality control scheme in place to monitor variation over time.

Boston University filed a patent (granted in 2010 in USA) on measuring anti-PLA2R for diagnosis of Membranous Nephropathy. A version of this original patent has been filed and granted in Europe as of Nov 2015. Euroimmune have the exclusive global licence to this patent and offer an ELISA kit and an immunofluorescence test kit for anti-PLA2R antibodies:

Euroimmun
AG D-23560
Luebeck
Germany

In UK, this Euroimmune ELISA assay is offered by the Protein Reference Unit in Sheffield:

Protein Reference Unit
Laboratory Medicine Building
North Lane
Northern General Hospital
Herries Road
Sheffield
S5 7AU
Tel: 0114 226 9196
Email: sht-tr.enquiriesPRUsheffield@nhs.net
www.immqas.org.uk

Common Questions

How specific is anti-PLA2R for MN?

It is highly specific for primary MN (>99%) but only found in up to 75% of cases naïve to immunosuppression. It is found in some cases (5-30%) of secondary MN (tumours, SLE) but this figure is based on small numbers of cases at present.

Can the anti-PLA2R test replace the need for biopsy?

No. Up to 25% of primary MN cases and 70-95% of secondary MN will be seronegative for anti-PLA2R whereas a biopsy will provide the pathological diagnosis of MN. Other tests using biopsy tissue are being developed and tested in research e.g. staining the kidney biopsy for the presence of PLA2R antigen in the immune complexes. This looks like a promising approach to confirm that the case involves PLA2R even though the patient may be seronegative at the time of biopsy. If confirmed in larger studies, archival biopsy tissue may confirm retrospectively the PLA2R status of a patient. Furthermore, the biopsy may also indicate features of secondary MN (with mesangial expansion, pan Igs and complement). There is some evidence suggesting high IgG1 and IgG3 and low IgG4 subclass ratio on biopsy may indicate secondary MN whereas the reverse pattern with high IgG4 is linked with primary MN. Renal biopsy will remain an important part of diagnosis of MN for several years yet.

How can a patient be biopsy positive for PLA2R but seronegative for anti-PLA2R?

A possible explanation is related to the affinity of the anti-PLA2R for its target antigen. We have recently presented work at the ASN (November 2013) describing anti-PLA2R antibodies as very high affinity antibodies (approx. 5 times higher than anti-GBM antibodies for their target antigen). This suggests that as anti-PLA2R antibodies are secreted by plasma cells and gain access to the circulation, they will be sequestered in the kidney, forming in situ immune complexes on podocytes. Only when the rate of antibody production exceeds the capacity of the kidney to adsorb the anti-PLA2R, is the patient likely to become seropositive.

I have a patient newly presenting with nephrotic syndrome and the renal biopsy is MN. The lab test for anti-PLA2R has come back very positive >3000 units/ml. Should I consider treating this patient with high levels of anti-PLA2R with a course of immunosuppression?

The clinical management of MN over the last 50 years has sought to define the benefit of immunosuppressive treatment versus the risks of comorbidities. When to treat, with what drug and for how long remain controversial, with few adequately powered, double blind randomised controlled trials to guide decisions. The conservative view would be to manage blood pressure and in the absence of evidence of decline in renal function, observe the patient over 6-9 months for evidence of spontaneous remission of proteinuria. Does knowledge that the patient has a high level of anti-PLA2R alter this conservative view?

Future Research

The most important question about anti-PLA2R that remains unanswered is ‘does anti-PLA2R cause proteinuria’ i.e is it pathogenic and driving the MN pathology as anti-GBM antibodies do in anti-GBM disease?

Currently, most of the data being generated from clinical studies relies on retrospective serum analysis of relatively small cohorts where samples have been biobanked and outcome data has been collected. These studies report:

  1. The association of high anti-PLA2R levels with increased risk of declining renal function and less chance of spontaneous remission over 5 years
  2. The association of high anti-PLA2R levels with active disease, low to normal values in remission and return to high levels of anti-PLA2R in relapse

Only in the last year have large prospective clinical trials been established using anti-PLA2R monitoring. These studies will take several years to confirm with confidence the role of anti-PLA2R. Similarly, the role of anti-PLA2R in recurrent MN post transplantation is actively being investigated as an agent causing recurrence. In a similar way to defining the pathogenicity of anti-GBM antibodies, it may be possible to establish active and passive experimental models of anti-PLA2R induced MN where cause and effect can be determined.

If pathogenicity of anti-PLA2R can be proven, this information is likely to influence the current conservative approach to treatment. Further work is required to establish if early intervention to remove anti-PLA2R is of benefit in improving outcomes, if tailoring treatment to remove anti-PLA2R results in fewer relapses and if ensuring MN patients are seronegative prior to transplantation reduces recurrent MN. These studies will take several years to complete and in the meantime, it is suggested that conservative management on when to treat is retained. Anti-PLA2R monitoring to indicate that immunosuppression is taking effect seems worthwhile as discussed in a recent editorial on use of anti-PLA2R monitoring for patient management (Glassock, 2014)

Reference List

  1. Beck LH, Jr., Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins TD, Klein JB, Salant DJ: M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 361:11-21, 2009.
  2. Stanescu HC, Arcos-Burgos M, Medlar A, Bockenhauer D, Kottgen A, Dragomirescu L, Voinescu C, Patel N, Pearce K, Hubank M, Stephens HA, Laundy V, Padmanabhan S, Zawadzka A, Hofstra JM, Coenen MJ, den HM, Kiemeney LA, Bacq-Daian D, Stengel B, Powis SH, Brenchley P, Feehally J, Rees AJ, Debiec H, Wetzels JF, Ronco P, Mathieson PW, Kleta R: Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy. N Engl J Med 364:616-626, 2011.
  3. Kanigicherla D, Gummadova J, McKenzie EA, Roberts SA, Harris S, Nikam M, Poulton K, McWilliam L, Short CD, Venning M, Brenchley PE: Anti-PLA2R antibodies measured by ELISA predict long-term outcome in a prevalent population of patients with idiopathic membranous nephropathy. Kidney Int 83:940-948, 2013.
  4. Fresquet M, Jowitt TA, Gummadova J, Collins R, O’Cualain R, McKenzie EA, Lennon R, Brenchley PE: Identification of a Major Epitope Recognized by PLA2R Autoantibodies in Primary Membranous Nephropathy. J Am Soc Nephrol, 2014 Oct 6 pii: ASN2014050502. PMID:25288605.
  5. Glassock RJ: Antiphospholipase A2 receptor autoantibody guided diagnosis and treatment of membranous nephropathy: a new personalized medical approach. Clin J Am Soc Nephrol 9:1341-1343, 2014.

Anti-PLA2R Assay Guidance Version 4 Updated July 2016
Written by Paul Brenchley on behalf of the Membranous Nephropathy Rare Disease Group