Can use of the serum anti-PLA2R antibody negate the need for a renal biopsy in primary membranous nephropathy?

Omar Ragy; Vilma Rautemaa; Alison Smith; Paul Brenchley; Durga Kanigicherla; Patrick Hamilton

doi:10.1371/journal.pone.0281726

. 2023 Feb 24;18(2):e0281726. doi: 10.1371/journal.pone.0281726

Can use of the serum anti-PLA₂R antibody negate the need for a renal biopsy in primary membranous nephropathy?

Omar Ragy ^1,^2,^☯,^*, Vilma Rautemaa ^1,^☯, Alison Smith ³, Paul Brenchley ^2,⁴, Durga Kanigicherla ^1,², Patrick Hamilton ^1,^2,⁴

Editor: Fabio Sallustio⁵

PMCID: PMC9955960 PMID: 36827283

Abstract

Background

Since the emergence of the anti-PLA₂R antibody (PLA₂R-Ab) test, nephrology practice has not changed dramatically, with most nephrologists still relying on a kidney biopsy to diagnose membranous nephropathy. In this study, we examined the clinical accuracy of the anti-PLA₂R antibody test using ELISA in routine clinical care.

Methods

We conducted a retrospective analysis of PLA₂R-Ab testing in 187 consecutive patients seen at a single UK centre between 2003 and 2020. We compared the kidney biopsy findings with the PLA₂R-ab antibody test. Patients’ demography, urine protein creatinine ratios, serum albumin, and treatment characteristics including supportive and immunosuppressive treatment were recorded. The clinical accuracy of the test (e.g. sensitivity and specificity, positive [PPV] and negative [NPV] predictive values) was calculated using the kidney biopsy findings as the diagnostic reference.

Results

Mean levels of PLA₂R-Ab titre in primary membranous nephropathy were 217RU/ml in comparison to 3RU/ml for both secondary membranous nephropathy and other diagnoses. Most patients with a positive PLA₂R-Ab test had a confirmed renal biopsy diagnosis of primary membranous nephropathy with: PPV of 97.3%, sensitivity 75.5%, NPV was 79.8% and specificity was 97.8% at a cut-off threshold of >20 RU/ml.

Conclusion

The anti-PLA₂R antibody test is a highly specific test for diagnosing membranous nephropathy, and the test has the potential to allow for the diagnosis and treatment in up to 75% of PMN cases without the need for a renal biopsy. Nevertheless, patients with negative PLA2R-Ab tests will still require a biopsy to confirm their diagnosis.

1. Introduction

Membranous nephropathy (MN) is one of the leading causes of nephrotic syndrome in adults worldwide [1]. Some cases can be secondary to malignancy, systemic autoimmune disease, drugs, or infections, but in the majority (~80%), it is a primary autoimmune disease caused by antibodies targeting the podocytes. The most common target autoantigen is the M-type phospholipase A2 receptor 1 (PLA₂R), identified by Beck et al in a pivotal study in 2009, found in up to 70% of patients with primary MN (PMN) [2]. Serum anti-PLA₂R autoantibody (PLA₂R-Ab) titres have shown promise as a highly specific diagnostic and prognostic biomarker [3, 4], and circumstantial evidence suggests the antibody is pathogenic. High titres are associated with active disease, a higher probability of disease relapse and a lower risk of remission following treatment. The converse is equally true with low titres associated with a higher probability of remission meaning its use as a biomarker for disease monitoring is particularly valuable [5–7].

Currently, clinical practice hinges on a renal biopsy to aid or confirm a diagnosis of primary MN (PMN). The wide availability and high specificity and sensitivity of the serum PLA₂R-Ab test has led many clinicians to start considering the use of the PLA₂R-Ab test as a diagnostic tool, particularly with the available evidence suggesting that amongst patients with positive PLA₂R-Ab test and preserved renal function, a renal biopsy may not provide significant additional information that would alter management. However, evidence is limited and currently based on small numbers [8], with further evidence required to convince nephrologist to alter their routine clinical practice.

2. Methods

2.1 Data collection

All patients, over the age of 18, with a renal biopsy and a PLA₂R-Ab test requested at the Manchester Royal Infirmary between January 2003 and January 2020 were eligible for the study. Samples were excluded if there was no reported PLA₂R-Ab test result, or if the antibody test was collected more than 6 months post-renal biopsy. If there were multiple samples from the same patient, the antibody result closest to the biopsy date was chosen and the other results were excluded. Patient demographics, renal biopsy findings, urine protein: creatinine ratio (UPCR) and serum albumin at the time of the PLA₂R-Ab test, use of immunosuppressants and renin-angiotensin system inhibitors (RAASi) were also recorded. Medical notes were reviewed by two independent clinicians to identify secondary causes of MN.

From 2017 onwards, all PLA2R-Ab tests were performed by the Sheffield Protein Reference Unit using the Euroimmun ELISA kit with levels <14 RU/ml interpreted as negative, 14–20 RU/ml as borderline, and >20 RU/ml as positive. For samples taken prior to 2017, all anti-PLA₂R antibody ELISA tests were performed using the in-house Manchester ELISA as described previously [7].

2.2 Ethics

After discussion with the Manchester University Hospitals NHS Foundation Trust Research and Innovation department), the study was exempt from requiring a specific ethical approval as it was considered a retrospective audit as per the Health Research Authority definition. The ethics committee therefore waived the requirement for informed consent and no consent or regulatory research approvals were required or obtained prior to this project commencing.

2.3 Statistical analysis

Statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software Inc, San Diego, CA). Statistical significance was set at 0.05.

Normality checked using shapiro-wilk test, and as all parameters significantly deviated from a normal distribution, continuous data are presented as median (interquartile range) and categorical data presented as number and percentage.

The receiver operating characteristic (ROC) curve was generated using established methods and the diagnostic sensitivity and specificity of the anti-PLA2R antibody test were calculated, using renal biopsy as the diagnostic reference standard [9–13]. 95% confidence intervals (CI) calculated using Wilson procedure with correction for continuity (Website http://vassarstats.net/prop1.html) [14, 15].

For analysis, all antibody titres reported as <3 or >3000 RU/ml were converted to 3 and 3000 RU/ml, respectively.

3. Results

3.1 Patient characteristics

In total, 187 adult patients who had an anti-PLA₂R antibody test before, or within 6 months after a renal biopsy, were included in the study (Fig 1). This included 94 patients with PMN, 7 with secondary MN (SMN), and 86 with other diagnoses. The most common other diagnoses for patients who had an PLA₂R-Ab test were minimal change disease, focal segmental glomerulosclerosis, IgA nephropathy, diabetes, and hypertension (Table 1). A significantly higher proportion of patients with PMN were male compared to patients with other diagnoses (72% vs 57% respectively, p<0.05).

Table 1. These are the patients’ demographics.

Continuous data are presented as median (range). UPCR: urine protein creatinine ratio, RAS: Renin Angiotensin System.

	Total	PMN	SMN	Other
Patients (n)	187	94	7	86
Age at PLA2R test	56 (18–88)	58 (23–86)	50 (18–75)	54 (18–88)
Gender (male)	122 (65%)	68 (72%)	5 (71%)	49 (57%)
Ethnicity (white)	143 (76%)	77 (82%)	5 (71%)	61 (71%)
PLA2R Ab Positive	73 (39%)	71 (76%)	0 (0%)	2 (2%)
UPCR at PLA2R test (mg/mmol)	603 (8–3976)	768 (8–2390)	435 (92–3976)	481 (31–2514)
Serum albumin at PLA2R test (g/L)	21 (6–43)	19 (6–42)	18 (7–34)	27 (7–43)
RAS inhibition	137 (73%)	85 (90%)	6 (85%)	46 (53%)
Immunosuppression	109 (58%)	73 (78%)	5 (71%)	31 (36%)

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3.2 PLA₂R antibody titres

The median antibody titre for patients with PMN was 219 (3–3000), with SMN 3 (3–12), and for other diagnoses 3 (2–36) RU/ml (Fig 2). Of 94 patients with PMN, 71 (76%) had a positive result. One patient with diffuse proliferative glomerulonephritis had an antibody titre of 31 RU/ml, and one patient with focal segmental glomerulosclerosis had a titre of 36 RU/ml. Apart from these two patients, nobody with a diagnosis other than PMN had a positive antibody test result.

3.3 Diagnostic sensitivity and specificity

The specificity of the anti-PLA₂R ELISA was 0.978 with a sensitivity of 0.755, whilst the Positive predictive value (PPV) was 97.3% and the negative predictive value (NPV) was 79.8% (Table 2). The PLA₂R-AB test has high specificity at the current threshold of 20 RU/ml (98%), however, the sensitivity is lower at 75%. Youden’s J statistic was highest at titre >10 RU/ml, but at levels >40 RU/ml specificity was 1 (Fig 3 and Table 3).

Table 2. This is the 2*2 diagnostic accuracy table.

Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the anti-PLA₂R ELISA with antibody titer ≥20 RU/ml interpreted as positive.

		Confirmed diagnosis
		Primary (PMN)	Not PMN (i.e. SMN or other)
Anti-PLA2R Test result	Positive	71	2	PPV
				71/73 = 0.973
				(95% CI 0.896–0.995)
	Negative	23	91	NPV
				91/114 = 0.798
				(95% CI 0.711–0.865)
		Sensitivity	Specificity
		71/94 = 0.755	91/93 = 0.979
		(95% CI 0.654–0.836)	(95% CI 0.917–0.996)

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Fig 3 — It shows the sensitivity and specificity of the serum PLA2R antibody test in our study. Area under curve: 0.943, p<0.0001.

Table 3. Sensitivity, specificity and Youden’s index of the anti-PLA2R test in diagnosing PMN in this study, over varying cut-off thresholds.

Ab Titer (RU/ml)	Sensitivity	Specificity	Youden’s J
>10	0.883	0.924	0.808
>20	0.756	0.978	0.734
>30	0.734	0.978	0.713
>40	0.691	1	0.691

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3.4 Renal function and medication

At the time of the PLA₂R-Ab test, patients with PMN had significantly higher uPCR and lower serum albumin than patients with other diagnoses (p<0.001 for both) (Table 1). During their treatment, 90% of patients with PMN received RAASi, compared to 53% of patients with other diagnoses (p<0.001). The use of immunosuppressants was also significantly higher in the PMN group, at 78%, compared to 36% of patients with other diagnoses (p<0.001). Patients with PMN who received immunosuppressants had significantly higher antibody titres than patients who did not receive immunosuppressant treatment (median 361 vs 42 RU/ml respectively, p<0.001). There was no significant correlation between antibody titre and renal function, UPCR and serum albumin at the time of biopsy in the PMN group.

4. Discussion

Current management of patients with suspected MN has changed little over the last 20 years. The vast majority of patients undergo a diagnostic renal biopsy with or without further screening investigations. With the discovery of the anti-PLA₂R antibody and the increasing understanding of its role in the disease pathogenesis, a more pragmatic patient-focused pathway becomes eminently possible. Our results show that the PLA₂R-Ab test had a specificity of 97.8% and sensitivity 76% for biopsy-confirmed PMN, with a titre of >20 RU/ml considered positive. This is in line with a previous meta-analysis from 2014 that found 99% specificity and 78% sensitivity for PMN [16]. The high specificity of the test suggests that we can rely solely on a positive test to diagnose and treat PMN without the need to perform a renal biopsy [17].

Despite its relative safety, an invasive procedure such as a renal biopsy comes with inherent risk of complications, in particular bleeding. The risk of bleeding is raised in those undergoing an emergency biopsy compared to an elective one [18]. Another important consideration is the cost of performing a biopsy. In England the national average unit cost of performing a renal biopsy in the NHS is £774, compared to a cost of £27 for an ELISA PLA₂R-Ab test [19]. This does not include any downstream costs brought on by complications, or societal costs such as taking time off work for the procedure, or those incurred by carers. The ability to safely avoid a biopsy in even a proportion of patients with MN therefore has implications for not only a patient’s quality of life but also healthcare systems in general.

One of the main disadvantages of PLA₂R-Ab is its low sensitivity. In our study 23 (24%) patients with biopsy-confirmed PMN had a negative PLA₂R-Ab test. This means that although the PLA₂R-Ab test is very good at ruling in PMN, a negative result cannot be used to rule out PMN as a diagnosis, and renal biopsy is indicated in these patients. There are several reasons why a patient with PMN can test negative for PLA₂R-Ab. Firstly, the patient may be in immunological remission, whether spontaneously or due to treatment [20]. Secondly, given the high affinity the anti-PLA₂R antibody has for the antigen, seronegativity in clinically active disease states can be attributed to an immune sink phenomenon [21]. Here, the antigen needs to be saturated with immune-complex deposition before there is a detectable circulating level [22]. In these situations, there is some evidence to show staining for the PLA₂R antigen on kidney biopsy may be of benefit in seronegative patients [23]. Thrombospondin 7A antigen (THSD7A) was found to be the target in 1–5% of PMN. Even more recently, Exostosin 1 and 2 were found to be target antigens in primary membranous nephropathy [24]. This makes the diagnosis of membranous nephropathy with a negative PLA₂R-Ab test still potentially valid, and in these situations, a biopsy warranted.

At present there are two generally available methods of assessing serum PLA₂R-Ab: immunofluorescence test (IFT) and the Euroimmun ELISA test [25]. Studies have shown a high concordance between these two tests with high specificity for PMN, especially when used together [8, 26, 27]. The ELISA test provides a quantitative result that can be used to guide treatment, whereas IFT is only semiquantitative but more sensitive at low antibody titres [20]. ELISA and IFT can be used together to increase diagnostic accuracy at low antibody titres and could be useful for patients where there is a high clinical suspicion of PMN but low or negative PLA₂R-Ab test.

Studies have shown that some patients with SMN can also have positive PLA₂R-Ab, especially SMN associated with hepatitis B infection [28]. This should be taken into account when diagnosing patients using the PLA₂R-Ab test, and screening for secondary causes of MN should be carried out as clinically indicated. However, in this study all patients with SMN had a negative PLA₂R-Ab result, indicating it is highly specific for PMN.

Anti-PLA₂R antibody levels have been shown to correlate with disease severity and clinical response. Patients with very high antibody titres have been found to have more significant reduction in renal function and lower remission rate than patients with lower titres [6, 29].

This study has a number of limitations. This was a retrospective cohort study, so data collection was limited to what was available on electronic patient records. Variability in the timing of biopsies and antibody testing, and other blood tests, has made the interpretation of the results more difficult. Many patients included in the study had their biopsy and PLA₂R-Ab test on different days, sometimes months apart. We have included renal function tests done on the day of the biopsy, which makes it difficult to correlate PLA₂R-Ab levels when performed at a different timing. A prospective study would be ideal to combat these limitations, however not practical due to the low incidence rate of the disease.

5. Conclusion

We have shown that a positive serum anti-PLA₂R antibody test has high specificity for PMN. This has the potential to allow for the diagnosis and treatment in up to 75% of PMN cases without the need for a renal biopsy. Patients with negative PLA₂R-Ab tests will still require a biopsy to confirm their diagnosis, and further screening for secondary causes of MN should also be considered in specific cases. But in a subset of patients, the ability to avoid an invasive procedure can provide a benefit to the patients journey and allow for significant cost savings to healthcare providers.

Data Availability

Data restrictions has been imposed by Manchester Foundation Trust Research and Innovation department. Data can be requested through our research and innovation department in Manchester Foundation trust (R&D.Applications@mft.nhs.uk).

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0281726.r001

Decision Letter 0

Fabio Sallustio

14 Feb 2022

PONE-D-21-33835Can Use of the serum anti-PLA2R antibody negate the need for a renal biopsy in Primary Membranous Nephropathy?PLOS ONE

Dear Dr. Ragy,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please, espond accurately to all the issues of the reviewers. Since the study is limited by the variability in the timing of biopsies and of antibody testing and by nuance in individual biopsies of MN that may impact accuracy of serum based testing, the conclusion of the paper should be less categorical but focusing to the importance of the results in the proceedings of MN diagnosis.

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Additional Editor Comments:

Since the study is limited by the variability in the timing of biopsies and of antibody testing and by nuance in individual biopsies of MN that may impact accuracy of serum based testing, the conclusion of the paper should be less categorical but focusing to the importance of the results in the proceedings of MN diagnosis.

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2. Has the statistical analysis been performed appropriately and rigorously?

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Interesting paper

Some issues.

Methods: due to reduced sample size, it should be added if normal distribution was checked for

Methods: was sample sixe calculation performed

Methods; was diagnosis at biopsy confitmed among different operators?

methods: do authors think thanks these results may be of clinical utility?

discussion; do authors think they enrolled enough patients?

Reviewer #2: Thank you for this opportunity to review your original research. I wholeheartedly agree with the authors that medical renal biopsy is an invasive procedure and carries inherent risk of adverse outcome. Thus, non-invasive study is ideal for patients who are poor candidates of biopsy.

In your study of serum PLA2R-Ab testing as potential replacement of biopsy, these are points that need clarification or would benefit from consideration:

1) Please elaborate on how primary MN (PMN) is defined. Patient's records were reviewed by nephrologists but did each patient have complete workup to exclude potential causes of secondary MN? Were histopathologic features of kidney biopsy considered (e.g. immunofluorescence staining pattern, location(s) of immune deposits, presence of other concomitant renal lesions)?

2) Any data on concordance rate of serum PLA2R-ab vs. tissue PLA2R staining on biopsy specimen? Any cases exhibiting "immune sink" phenomenon?

3) Renal biopsies are obtained for both diagnostic and prognostic purposes. Thus, it would be worthwhile to investigate the performance of serum PLA2R-Ab while considering other parameters (e.g. %global and segmental glomerulosclerosis, % tubulointerstitial scarring, tubulointerstitial inflammation, Ehrenreich-Churg stage of MN)

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Reviewer #1: Yes: Fabrizio D'Ascenzo

Reviewer #2: No

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PLoS One. 2023 Feb 24;18(2):e0281726. doi: 10.1371/journal.pone.0281726.r002

Author response to Decision Letter 0

27 Apr 2022

First reviewer’s questions:

1) Methods: due to reduced sample size, it should be added if normal distribution was checked for

Added this sentence in the statistical analysis section:

2) Methods: was sample size calculation performed?

As this was a retrospective single centre analysis, the sample size was determined by the data we had available to us. We have not conducted any hypothesis tests, but we have added in 95% confidence interval estimates to clearly convey the uncertainty around our estimates.

The manuscript has been updated to reflect this. The receiver operating characteristic (ROC) curve was generated using established methods and the diagnostic sensitivity and specificity of the anti-PLA2R antibody test were calculated, using renal biopsy as the diagnostic reference standard (9,10,11,12,13). 95% confidence intervals (CI) calculated using Wilson procedure without correction for continuity

Ref – Website http://vassarstats.net/prop1.html accessed on 29/03/22

And the references from the website added:

Newcombe, Robert G. "Two-Sided Confidence Intervals for the Single Proportion: Comparison of Seven Methods," Statistics in Medicine, 17, 857-872 (1998).

Wilson, E. B. "Probable Inference, the Law of Succession, and Statistical Inference," Journal of the American Statistical Association, 22, 209-212 (1927).

3) Methods: was diagnosis at biopsy confirmed among different operators?

All biopsies were reported by a Renal Histopathologist and discussed at the departmental biopsy review MDT.

4) Methods: do authors think thanks these results may be of clinical utility?

We believe that those results will be a steppingstone to move away from performing renal biopsies for patients with a positive PLA2R ab test

5) Discussion: do authors think they enrolled enough patients?

As far as we are aware, this is the largest retrospective study to address that question. Given that MN is listed on the National Registry of Rare Kidney Disease (RaDaR), so we think that the number of patients enrolled to answer our question was enough

Second reviewer’s questions:

1)Please elaborate on how primary MN (PMN) is defined?

PMN was confirmed based on a renal biopsy, having a positive anti PLA2R ab test and negative secondary work up.

2)Patient's records were reviewed by nephrologists but did each patient have complete workup to exclude potential causes of secondary MN?

Secondary workup for cancers was only performed for certain high-risk categories (older than 50 years old, smokers, history of change in bowel habits and signs of iron deficiency anaemia), atypical findings on histology or based on full history and examination. PSA for those >50 years old and CT TAP/colonoscopy and virology screen were done only when clinically indicated. Autoimmune screen was only performed if other autoimmune conditions were present. The most recent evidence from KDIGO 2021 is still to perform a secondary work up regardless the antibody test result.

3)Were histopathologic features of kidney biopsy considered (e.g. immunofluorescence staining pattern, location(s) of immune deposits, presence of other concomitant renal lesions)?

Our findings were not specifically correlated with histological features on biopsy other than the diagnosis of MN itself and we did not stain for PLA2R1 antigen on the renal tissue

4) Any data on concordance rate of serum PLA2R-ab vs. tissue PLA2R staining on biopsy specimen? Any cases exhibiting "immune sink" phenomenon?

We did not stain for PLA2R1 antigen on the renal tissue to correlate that with serum antibody test.

5) Renal biopsies are obtained for both diagnostic and prognostic purposes. Thus, it would be worthwhile to investigate the performance of serum PLA2R-Ab while considering other parameters (e.g. %global and segmental glomerulosclerosis, % tubulointerstitial scarring, tubulointerstitial inflammation, Ehrenreich-Churg stage of MN)

This would be beyond the scope of our study. We agree that this might be of useful prognostic value, but not for diagnostic purposes. Our study was mainly focusing on the diagnostic accuracy of the antibody test.

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PLoS One. doi: 10.1371/journal.pone.0281726.r003

Decision Letter 1

Fabio Sallustio

1 Jul 2022

PONE-D-21-33835R1Can Use of the serum anti-PLA2R antibody negate the need for a renal biopsy in Primary Membranous Nephropathy?

PLOS ONE

Dear Dr. Ragy,

In the light of the Reviewer 2 comments we believe that at least a subset of samples should be stained for PLA2R1 antigen on the renal tissue to correlate that with serum antibody test. A correlation with histopathologic features of kidney biopsy should be considered, as well.

Please submit your revised manuscript by Aug 15 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Fabio Sallustio, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: (No Response)

Reviewer #2: No

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: I am disappointed to learn that pathologic parameters from the biopsy were not considered in your study to determine if serum antiPLA2R Ab test should replace kidney biopsy in diagnosis of MN. While PLA2R+ MN is most often a PMN, other investigators have reported cases of PLA2R+ MN associated with HBV infection (Am J Nephrol 2015;41(4-5):345-53.) and PLA2R+ MN associated with malignancy (Clin Kidney J 2015; 8: 433-9). Moreover, there is also report of low prevalence serum antiPLA2R ab in patients with membranous lupus nephritis (Lupus. 2019 Mar;28:396-4050). Thus, I am in disagreement with the author's conclusions that "diagnosis of primary membranous nephropathy can rely solely on a positive PLA2 R-Ab test without the need for renal biopsy"

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Fabrizio D'Ascenzo

Reviewer #2: No

**********

PLoS One. 2023 Feb 24;18(2):e0281726. doi: 10.1371/journal.pone.0281726.r004

Author response to Decision Letter 1

8 Jan 2023

Dear PLOS, ONE reviewer

Many thanks for the time given to review our work and for providing us with your valuable feedback.

The utility and validity of pathological parameters in kidney biopsy tissue is a salient research question, including PLA2R staining; others including IgG4 vs staining IgG1 staining (1), neutrophils in glomeruli (2). We would however wish to note that staining PLA2R1 for histopathological samples to correlate with serum antibody test is beyond the scope of our study, and we believe it will not necessarily answer the primary question on the diagnostic utility of serum PLA2R-Ab testing. We hypothesize that the serum PLA2R-Ab test can replace the gold standard renal biopsy (using conventional stains, with PLA2R staining being unavailable or yet to be standardised in many centres). The objective of our study was to compare the reference test (standard renal biopsy) without PLA2R stain vs the index test (serum PLA2R-Ab test that is readily available as a commercial test) to determine if serum PLA2R-Ab testing can replace the renal biopsy.

Whilst we agree with reviewer #2 that in certain situations, the PLA2R-ab test can be falsely positive and that a tissue diagnosis may provide more insight, staining for PLA2R1 in addition to serum testing will only likely improve the sensitivity with diagnosis. This question was studied by other investigators in previous investigations. Larsen et al, 2013, examined PLA2R1 in renal tissues in 165 cases of MN, including 85 primary MN and 80 secondary MN, and found tissue staining to have a sensitivity of 75% and a specificity of 83% (3). Hill et al, found that combining both biopsy and serum testing only improved the diagnostic sensitivity of the test, but not the specificity. In the aforementioned study, the specificity of the PLA2R-Ab test alone was 100% and the sensitivity was 81% compared to 100% and 89.5% respectively for tissue staining. When they combined both tissue and serum testing, the sensitivity improved to 95.2%, but the specificity was already 100% without the need for a biopsy (5). Therefore, we do agree that a biopsy in situations where the PLA2R-Ab test was negative remains an indispensable test, but not when the anti-PLA2R antibody test in serum were to be positive. In our current study, only 2 subjects had a false positive result out of 93 subjects, with a specificity of 97.9%. Even the presence of tissue staining for PLA2R or IgG4 can only raise the possibility that such cases are pathogenically related to primary MN than secondary MN (3). Many other investigators noted the similar association, especially with Hepatitis B and Sarcoidosis.

Debiec and Ronco et al, have shown that some patients may have positive serum PLA2R- Ab test and their antigen stain on biopsy is negative, which is another challenging question on why the biopsy stain would not correlate with serum testing (6). We would argue that the complexity of the immune system and antigenicity of the podocyte is not fully discovered, and you would agree with us that in those situations even if a biopsy was performed, it might not change the clinician’s decision to treat.

Also, one would appreciate that even the renal biopsy being the gold standard diagnostic test for many years, it will not always provide 100% specificity and in some situations, it might miss a true diagnosis due to various reasons. Therefore, tissue or serum positivity would only suggest PLA2R association, but not being a primary or secondary driven pathology (4). Differentiation between Primary and Secondary may still require standard evaluation with clinical history, examination and screening for secondary causes.

We have reviewed the papers you have cited for our attention, which state that HBV, SLE, and cancers can result in a falsely positive PLA2R-Ab test. This observation is true, but only reported in a small number of studies. Performing a renal biopsy for those small number of patients needs to be weighed against the other larger population who could potentially avoid renal biopsy with its downstream costs (e.g., relating to complications including additional inpatient stays in hospital, blood transfusions, interventions post-bleeding complications), and the societal cost of biopsy (e.g., healthcare cost, patients and carer’s taking time off work). Moreover, this directs us to highlight that all patients who present with nephrotic syndrome, will have had a routine screening for HBV and SLE at disease presentation, which may already underpin the diagnosis of those conditions even before the PLA2R-Ab test results are available.

Having both HBV serology and PLA2R-Ab test positive, one may consider either a diagnosis of Primary membranous nephropathy (PMN) complicated by HBV or secondary membranous nephropathy (SMN) due to HBV (7). In this scenario, a renal biopsy can only aid the diagnosis if PLA2R1, c1q, and IgG subclasses can be stained, which is not widely available in clinical practice. Hence, a routine renal biopsy without the use of those special stains might not add value to the serum PLA2R-Ab test.

MN secondary to SLE has also been a challenging diagnosis even after the discovery of PLA2R1 antigen. Svobodovo et al, found that none of their 16 Czech patients with a biopsy confirmed diagnosis of SMN due to SLE had a PLA2R1 positive stain on the renal tissue (8). These findings strongly suggest that lupus MN is not always related to PLA2R-Abs. In that situation, although this might not be PLA2R1-driven pathogenesis, we would urge a renal biopsy given the negative serum PLA2R-Ab testing. In other scenarios where the serum PLA2R-Ab test was positive in a patient with SLE, we would highly recommend a secondary workup including ANA, anti-DNAs, and complement levels, which is clearly stated in our conclusion in manuscript line number 219 and 220 ‘further screening for secondary causes of MN should also be considered in specific cases’

We cannot ascertain that a renal biopsy would necessarily diagnose PLA2R-associated MN triggered by a secondary cause. Radice et al identified 7 cases of cancer-associated MN confirmed with biopsy. All had their serum PLA2R-Ab tested positive, yet none of those renal biopsies could help identify that malignancy can be the culprit in the MN diagnosis. (9).Lefaucheur et al used a cut-off of eight cells per glomerulus to distinguish malignancy-related MN cases from controls, they calculated the specificity as 75% and sensitivity as 92% with an area under the curve of 0.92(2). This would highlight that performing biopsy would not stand a better chance to diagnose malignancy compared to the serum PLA2R -Ab test sensitivity and specificity. To make the diagnosis of cancer-related MN even more challenging, in many situations MN diagnosis would precede the cancer diagnosis. Again, a biopsy in those scenarios would not add value to positive PLA2R-Ab serum testing. Nonetheless, cancer screening for the high-risk group would be urged as suggested in our conclusion.

We hope that through extensive literature review, we demonstrated that even if the serum PLA2R-Ab test was falsely positive in limited scenarios, a biopsy performed in clinical practice using the conventional stains might not add much value to the diagnosis. Also, we emphasize the importance of considering PMN with another independent disease like cancer, SLE, or HBV infection in situations where both the serum PLA2R-Ab and screening tests were positive.

References:

1. Huang, C. C., Lehman, A., Albawardi, A., Satoskar, A., Brodsky, S., Nadasdy, G., Hebert, L., Rovin, B., & Nadasdy, T. (2013). IgG subclass staining in renal biopsies with membranous glomerulonephritis indicates subclass switch during disease progression. Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc, 26(6), 799–805. https://doi.org/10.1038/modpathol.2012.237

2. Lefaucheur C, Stengel B, Nochy D, et al. Membranous nephropathy and cancer: Epidemiologic evidence and determinants of high-risk cancer association. Kidney Int. 2006;70(8):1510-1517. doi:10.1038/sj.ki.5001790

3. Larsen, C. P., Messias, N. C., Silva, F. G., Messias, E., & Walker, P. D. (2013). Determination of primary versus secondary membranous glomerulopathy utilizing phospholipase A2 receptor staining in renal biopsies. Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc, 26(5), 709–715. https://doi.org/10.1038/modpathol.2012.207

4. Disease, K. (2021). Kidney Disease: Improving Global Outcomes (KDIGO) Glomerular Diseases Work Group. KDIGO 2021 clinical practice guideline for the management of glomerular diseases. Kidney Int, 100(4S), S1–S276. https://doi.org/10.1016/j.kint.2021.05.021

5. Hill, P. A., McRae, J. L., & Dwyer, K. M. (2016). PLA2R and membranous nephropathy: A 3 year prospective Australian study: PLA2R and membranous nephropathy. Nephrology (Carlton, Vic.), 21(5), 397–403. https://doi.org/10.1111/nep.12624

6. Debiec, H., & Ronco, P. (2011). PLA2R autoantibodies and PLA2R glomerular deposits in membranous nephropathy. N Engl J Med, 364, 689–690)

7. Wang, R., Wu, Y., Zheng, B., Zhang, X., An, D., Guo, N., Wang, J., Guo, Y., & Tang, L. (2021). Clinicopathological characteristics and prognosis of hepatitis B associated membranous nephropathy and idiopathic membranous nephropathy complicated with hepatitis B virus infection. Scientific Reports, 11(1), 18407. https://doi.org/10.1038/s41598-021-98010-y

8. Svobodova, B., Honsova, E., Ronco, P., Tesar, V., & Debiec, H. (2013). Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy. Nephrology, Dialysis, Transplantation: Official Publication of the European Dialysis and Transplant Association - European Renal Association, 28(7), 1839–1844. https://doi.org/10.1093/ndt/gfs439

9. Radice, A., Pieruzzi, F., Trezzi, B., Ghiggeri, G., Napodano, P., D’Amico, M., Stellato, T., Brugnano, R., Ravera, F., Rolla, D., Pesce, G., Giovenzana, M. E., Londrino, F., Cantaluppi, V., Pregnolato, F., Volpi, A., Rombolà, G., Moroni, G., Ortisi, G., & Sinico, R. A. (2018). Diagnostic specificity of autoantibodies to M-type phospholipase A2 receptor (PLA2R) in differentiating idiopathic membranous nephropathy (IMN) from secondary forms and other glomerular diseases. Journal of Nephrology, 31(2), 271–278. https://doi.org/10.1007/s40620-017-0451-5

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PLoS One. doi: 10.1371/journal.pone.0281726.r005

Decision Letter 2

Fabio Sallustio

27 Jan 2023

PONE-D-21-33835R2Can Use of the serum anti-PLA2R antibody negate the need for a renal biopsy in Primary Membranous Nephropathy?PLOS ONE

Dear Dr. Ragy,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but it need minor revisions in the Abstract. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The Abstract conclusions are overstated and should be in line with that reported at the end of the paper. Please, revise the Abstract conclusions highlighting that antibody test has the potential to allow for the diagnosis and treatment in up to 75% of PMN cases without the need for a renal biopsy but that patients with negative PLA2R-Ab tests will still require a biopsy to confirm their diagnosis.

Please submit your revised manuscript by Mar 13 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Fabio Sallustio, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

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Reviewers' comments:

PLoS One. 2023 Feb 24;18(2):e0281726. doi: 10.1371/journal.pone.0281726.r006

Author response to Decision Letter 2

29 Jan 2023

Dear reviewer

Thank you for considering our work . We have reviewed your comments, and agree with your suggestions about changing the abstract conclusion. You will find the new version dated 29/1/23 amended accordingly.

Many thanks for your help

Omar Ragy

Consultant Nephrologist

Manchester Institute of Nephrology and Transplantation

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