NELL1 is a target antigen in malignancy-associated membranous nephropathy

Abstract

Patients with membranous nephropathy have an increased risk of malignancy compared to the general population, but the target antigen for malignancy-associated membranous nephropathy is unknown. To explore this, we utilized mass spectrometry for antigen discovery in malignancy-associated membranous nephropathy examining immune complexes eluted from frozen kidney biopsy tissue using protein G bead immunoglobulin capture. Antigen discovery was performed comparing cases of membranous nephropathy of unknown and known type. Mass spectrophotometric analysis revealed that nerve epidermal growth factor-like 1 (NELL1) immune complexes were uniquely present within the biopsy tissue in membranous nephropathy. Additional NELL1-positive cases were subsequently identified by immunofluorescence. In a consecutive series, 3.8% of PLA2R- and THSD7A-negative cases were NELL1-positive. These NELL1-positive cases had segmental to incomplete IgG capillary loop staining (93.4%) and dominant or co-dominant IgG1-subclass staining (95.5%). The mean age of patients with NELL1-positive membranous nephropathy was 66.8 years, with a slight male predominance (58.2%) and 33% had concurrent malignancy. Compared with PLA2R- and THSD7A-positive cases of membranous nephropathy, there was a greater proportion of cases with malignancies in the NELL1-associated group. Thus, NELL1-associated membranous nephropathy has a unique histopathology characterized by incomplete capillary loop staining, IgG1-predominance, and is more often associated with malignancy than other known types of membranous nephropathy. Patients with membranous nephropathy have an increased risk of malignancy compared to the general population, but the target antigen for malignancy-associated membranous nephropathy is unknown. To explore this, we utilized mass spectrometry for antigen discovery in malignancy-associated membranous nephropathy examining immune complexes eluted from frozen kidney biopsy tissue using protein G bead immunoglobulin capture. Antigen discovery was performed comparing cases of membranous nephropathy of unknown and known type. Mass spectrophotometric analysis revealed that nerve epidermal growth factor-like 1 (NELL1) immune complexes were uniquely present within the biopsy tissue in membranous nephropathy. Additional NELL1-positive cases were subsequently identified by immunofluorescence. In a consecutive series, 3.8% of PLA2R- and THSD7A-negative cases were NELL1-positive. These NELL1-positive cases had segmental to incomplete IgG capillary loop staining (93.4%) and dominant or co-dominant IgG1-subclass staining (95.5%). The mean age of patients with NELL1-positive membranous nephropathy was 66.8 years, with a slight male predominance (58.2%) and 33% had concurrent malignancy. Compared with PLA2R- and THSD7A-positive cases of membranous nephropathy, there was a greater proportion of cases with malignancies in the NELL1-associated group. Thus, NELL1-associated membranous nephropathy has a unique histopathology characterized by incomplete capillary loop staining, IgG1-predominance, and is more often associated with malignancy than other known types of membranous nephropathy. Membranous nephropathy (MN) is the most common cause of idiopathic nephrotic syndrome in adults. It is a disease caused by the deposition of immune complexes in a glomerular distribution. It can be caused by antibodies directed against podocyte antigens (primary MN) or through secondary deposition of circulating immune complexes (or alternatively in situ immune complex formation) in glomeruli. The most common causes of secondary MN are autoimmune diseases, of which systemic lupus erythematosus accounts for the majority of cases (membranous lupus nephritis). The second most common secondary etiology is malignancy. Other causes include infections (such as viral hepatitis), graft-versus-host disease, allergic responses (such as against bovine serum albumin), and drug reactions. MN is the most common glomerular disease occurring coincidently with neoplasms. It can be the first sign of an occult malignancy in up to 20% of cases.1Beck J.L. Membranous nephropathy and malignancy.Semin Nephrol. 2010; 30: 635-644Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar This association was first reported by Lee et al. in 1966, where 11% of patients with nephrotic syndrome had an underlying carcinoma, and enriched in patients with MN (73%).2Lee J.C. Yamauchi H. Hopper Jr., J. The association of cancer and the nephrotic syndrome.Ann Intern Med. 1966; 64: 41-51Crossref PubMed Scopus (298) Google Scholar The risk of cancer in patients with MN is 2- to 3-fold higher than the age-matched general population up to 4 years after the diagnosis of MN.3Birkeland S.A. Storm H.H. Glomerulonephritis and malignancy: a population-based analysis.Kidney Int. 2003; 63: 716-721Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Although malignant neoplasms are a known “secondary” association of MN, determining who should undergo a thorough workup to exclude a coexistent cancer is a subject of debate. Risk factors for concurrent malignancy include older age (>65 years), a ≥20-pack-year smoking history, use of chronic immunosuppression, or presence of thrombotic complications (such as renal vein thrombosis, deep venous thrombosis, or pulmonary embolism).4Pani A. Porta C. Cosmai L. et al.Glomerular diseases and cancer: evaluation of underlying malignancy.J Nephrol. 2016; 29: 143-152Crossref PubMed Scopus (30) Google Scholar In a study by Ronco, as many as 22% of patients with MN who were 60 years of age or older had malignancy, which was 10-fold higher than that in age-matched controls.5Ronco P.M. Paraneoplastic glomerulopathies: new insights into an old entity.Kidney Int. 1999; 56: 355-377Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Other factors characteristic of malignancy-associated MN include phospholipase A2 receptor (PLA2R) negativity and IgG1- and/or IgG2-restricted subclass predominance.6Lonnbro-Widgren J. Ebefors K. Molne J. et al.Glomerular IgG subclasses in idiopathic and malignancy-associated membranous nephropathy.Clin Kidney J. 2015; 8: 433-439Crossref PubMed Scopus (24) Google Scholar,7Ohtani H. Wakui H. Komatsuda A. et al.Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy.Nephrol Dial Transplant. 2004; 19: 574-579Crossref PubMed Scopus (144) Google Scholar Neural epidermal growth factor–like 1 (NELL1) is a recently described antigen in MN.8Sethi S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Our findings suggest that NELL1-associated MN is commonly associated with malignancy and has unique histopathologic findings that may aid in the identification of cases in everyday clinical practice. Protein G was used to isolate IgG-containing immune complexes from frozen tissue remnants from each of the 59 renal biopsies. These affinity-purified immune complexes were then analyzed by mass spectrometry (MS) to determine their proteomic composition. Two groups were compared: the first group contained MN cases of known antigen types, including phospholipase A2 receptor (PLA2R, n = 4), thrombospondin type-1 domain-containing 7A (THSD7A, n = 3), and exostosin 1/2 (EXT1/2, n = 4). Diabetic glomerulopathy and arterionephrosclerosis samples with no positive immunofluorescence staining were used as negative controls (n = 7). The second group contained cases negative for PLA2R, THSD7A, and EXT1/2 (n = 41). By comparing proteomes from the known with the unknown MN types, it was possible to discern unique proteins. This technique was highly sensitive and specific for the identification of antigenic targets in MN of known type. PLA2R was the only protein that immunoprecipitated with IgG in 4 of 4 PLA2R-positive MN biopsies and 0 of 55 remaining biopsies (Supplementary Figure S1A). THSD7A was the only protein identified in 3 of 3 THSD7A-positive MN cases and 0 of 56 remaining biopsies (Supplementary Figure S1B). EXT was identified in 4 of 4 EXT-positive MN cases and 0 of 55 remaining biopsies (Supplementary Figure S1C). There were no proteins that uniquely immunoprecipitated with IgG in diabetic nephropathy or arterionephrosclerosis samples. Thus, as a proof of principle, this approach specifically identified the autoantigen in cases with MN of known type. When the same analysis was applied to cases of MN of unknown antigenic type, there were 2 of 41 cases in which NELL1 was identified (Figure 1). No membranous lupus nephritis cases (which included 16 of 41 PLA2R-negative, THSD7A-negative, and EXT1/2-negative MN cases) submitted for MS were NELL1 positive. This analysis confirms that IgG is bound to NELL1 in a subset of MN cases, which were also confirmed to be positive by tissue immunofluorescence staining. Dual NELL1-Rhodamine Red-X (Jackson Immunoresearch, West Grove, PA) and IgG-fluorescein isothiocyanate (FITC) labeling showed near-complete colocalization of NELL1 with IgG by confocal microscopy (86.7% ± 9.4%) (Supplementary Figure S2, top panel). There was no significant colocalization of NELL1 and IgG in PLA2R-positive MN cases used as controls (25.1% ± 14.4%) (Supplementary Figure S2, bottom panel). A graphical representation of the colocalization data is provided in Supplementary Figure S3. There was a significant increase in NELL1/IgG colocalization in NELL1-positive MN cases compared with PLA2R-positive MN controls (P < .0001). To determine the frequency of NELL1 positivity in MN, we stained 349 consecutive MN cases with tissue available for PLA2R, THSD7A, EXT2, and NELL1. This series included 83 pure class V membranous lupus nephritis (no proliferation) and 266 MN the 349 cases, were PLA2R positive were THSD7A positive were positive and were NELL1 positive the 266 MN cases were PLA2R positive were THSD7A positive were positive and were NELL1 positive the 83 class V membranous lupus nephritis cases, 1 was PLA2R positive 3 were THSD7A positive were positive and 1 was NELL1 positive there were of a of cases from the consecutive series to clinical or of NELL1-associated MN, NELL1-associated MN cases were identified from the of years of MN In NELL1-associated MN cases were NELL1-associated MN cases showed glomerular capillary (Figure of cases showed the of or (Figure often in a segmental in glomeruli. A segmental of of glomerular to incomplete by capillary loop staining was in of cases of NELL1-associated MN (Figure and and in no cases of of or of capillary loop staining was in of NELL1-associated MN compared with of PLA2R-positive MN and of THSD7A-positive MN cases (Supplementary Figure The segmental of immune deposits was also by the of deposits by of glomerular capillary (Figure with other deposits (Figure of patients with NELL1-associated MN showed no significant in positivity NELL1 = positivity NELL1 = and positivity NELL1 = and a immunofluorescence with the of and NELL1 = as compared with PLA2R-positive MN 1). positivity was common in NELL1-associated MN than in PLA2R-positive MN NELL1 = There was no significant staining in cases of NELL1-associated MN, with no cases deposits or MN cases had a higher frequency of and and the presence of deposits than or THSD7A-positive MN A of the histopathologic is in of kidney biopsies with NELL1-associated membranous nephropathy compared with and membranous (n = (n = (n = (n = NELL1 capillary loop of of of of segmental of of of of or incomplete capillary loop of of of of of of of of of of of of of of of of of of of of of of of of of of of of of and deposits are from cases of cases, as 3 of cases have a available for evaluation by of of of of and deposits are from cases of cases, as 3 of cases have a available for evaluation by of of of nerve epidermal growth factor-like PLA2R, phospholipase A2 THSD7A, thrombospondin type-1 domain-containing include frequency and deposits are from cases of cases, as 3 of cases have a available for evaluation by in a new EXT2, nerve epidermal growth factor-like PLA2R, phospholipase A2 THSD7A, thrombospondin type-1 domain-containing include frequency IgG subclass staining was performed cases with frozen tissue available for There was staining in cases of NELL1-associated MN of cases) (Figure Supplementary Figure and of cases were dominant of cases had staining was present in of cases and concurrent staining was present in of cases of cases were and and of cases were and of NELL1-associated MN cases had dominant or subclass staining (Figure There were 3 of cases with and staining a in autoimmune subclass staining in NELL1-associated or greater is of of of of of of of of of of of of of of membranous epidermal growth factor–like or greater is in a new MN, membranous epidermal growth factor–like microscopy was available for of cases of NELL1-associated A majority of cases showed podocyte of cases with of cases podocyte and of cases podocyte NELL1-associated MN cases had a higher of deposits of cases for NELL1 of cases for PLA2R = and no deposits of cases 2 of cases = 1). Patients with NELL1-associated MN were older than patients with PLA2R-positive MN 66.8 ± years NELL1 ± years patients with MN ± years), and MN ± Patients with NELL1-associated MN with malignancy were older than patients malignancy ± years ± = There was a slight male predominance a of NELL1-associated MN cases had a of of only had in diabetic nephropathy of patients with NELL1-associated MN had and 2 of showed concurrent Additional disease comparing and NELL1-positive MN are provided in Supplementary Two cases of NELL1-associated MN had a of To explore this, we stained cases of and THSD7A-negative MN in patients with a of disease, or disease from and that 2 of cases were NELL1 positive. Although NELL1 was identified as a of in disease in a association A. J. et association NELL1 as a disease PubMed Scopus Google Scholar this a secondary etiology for the of NELL1-associated MN in a subset of patients is years of idiopathic MN biopsies from were to malignancy-associated MN malignancy-associated MN cases were of which were PLA2R 4 were THSD7A and were NELL1 positive the higher of PLA2R-positive MN than of NELL1-positive MN, patients with cancer are more to have PLA2R-positive the of malignancy is higher in patients with NELL1-positive MN than in with other types of 33% of NELL1-positive patients had malignancy of only of PLA2R-positive patients of and of THSD7A-positive patients had concurrent malignancy of Two THSD7A-positive patients and 4 NELL1-positive cases had 2 concurrent The associated antigens for malignancy-associated MN were unknown in of 3 had 2 (Supplementary Figure of malignancy in NELL1-associated MN compared with MN, MN, and MN to unknown types and of in patients with malignancy-associated MN are of (n = of (n = 4 of (n = of antigen (n = of 4 patients in the THSD7A-positive had a of malignancy, the of is of 2 patients with 2 patients in the NELL1-positive had a of malignancy, the of is of 4 patients with 2 patients with MN of unknown had a of malignancy, and the of is of 3 patients with 2 MN, membranous epidermal growth factor–like PLA2R, phospholipase A2 THSD7A, thrombospondin type-1 domain-containing The types and of in patients with malignancy-associated MN are Although 4 patients in the THSD7A-positive had a of malignancy, the of is of 2 patients with 2 Although patients in the NELL1-positive had a of malignancy, the of is of 4 patients with 2 patients with MN of unknown had a of malignancy, and the of is of 3 patients with 2 in a new MN, membranous epidermal growth factor–like PLA2R, phospholipase A2 THSD7A, thrombospondin type-1 domain-containing the of MN within this where PLA2R-positive MN up of cases of MN in but only in this to to the diagnosis of et for a approach to membranous Nephrol. 2016; PubMed Scopus Google Scholar with and available for of patients with nephrotic A majority of patients with NELL1-associated MN with a of malignancy had concurrent and malignancy (n = of patients had the cancer identified 1 had malignancy identified in a workup for causes of secondary and in 2 the underlying malignancy the diagnosis of MN but the of diagnosis was unknown (Supplementary of patients had disease the of patients in the diagnosis of malignancy was compared with the diagnosis of MN or in (n = 4 of NELL1-positive patients had a of patients in had The the diagnosis of cancer and MN in is in Supplementary the of biopsy and were in patients with NELL1-associated MN and with or MN PLA2R ± THSD7A ± NELL1 ± available for of PLA2R ± THSD7A ± NELL1 ± which data were available for 59 of A majority of patients in groups had renal the of with the to with concurrent kidney A majority of patients with NELL1-associated MN had nephrotic the of biopsy to of 59 patients with available were in of patients of patients with serum available with mean ± of the associated was available from 2 patients with NELL1-associated In 1 with NELL1-associated MN and concurrent of the NELL1 positivity by immunofluorescence and staining was in the biopsy (Supplementary Figure and and (Supplementary Figure and A with also NELL1 staining an biopsy (Supplementary Figure and patients with NELL1-associated MN had samples available for serum against the NELL1 of patients with NELL1-associated MN had serum to NELL1 protein in Supplementary Figure No from PLA2R-positive MN cases showed of 3 of NELL1-positive patients serum were in clinical with a in the of serum compared with the of with of MN) and data were available from 59 of patients In to 59 of patients in were as were or of and of for clinical after their The mean clinical was ± The was ± The in from the of biopsy was ± of 59 patients were in with of of 59 patients were in with to of or a in from the of of 59 patients had no with in and of A majority of patients had renal ± of the of were patients with available data (n = and were patients and/or receptor n = the the of biopsy was ± the was ± and the was ± In patients (n = the the of biopsy was ± the was ± and the was ± In patients with (n = 3), the the of biopsy was the was and the was provided of In patients with and (n = 3), the the of biopsy was the was and the was provided of was with who with of and had of and of patients of an underlying malignancy and concurrent (n = the was ± the was ± and the was ± of the of patients (n = an of NELL1-associated MN be patients with of an underlying malignancy showed to of patients for malignancy concurrent in or The and data after is included in Supplementary S3. Our study has clinical was available for patients with malignancy-associated MN can have cancer identified a workup after the diagnosis of their glomerular disease up to 2 to 4 years after MN and we have of patients for years, data the frequency of malignancy in patients with NELL1 is a which is in in but is where it is for and It to thrombospondin and of 1 3 1 thrombospondin type-1 and epidermal growth S. et and analysis of proteins NELL1 and 1999; PubMed Scopus Google Scholar PLA2R or THSD7A, a and has a et al. recently showed that NELL1-positive MN was a specific of MN and was the antigen in up to of MN S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar identified NELL1-associated MN in 3.8% of MN cases that were negative for PLA2R and association with malignancy was higher in compared with their malignancy association of The histopathology of NELL1-associated MN was unique compared with other of This segmental to incomplete IgG staining, subclass and of staining for other immune and of the histopathologic in NELL1-associated MN have described in of malignancy-associated MN in the These include and subclass PLA2R-positive MN cases more had deposits and malignancy-associated MN cases had a frequency of J. Ebefors K. Molne J. et al.Glomerular IgG subclasses in idiopathic and malignancy-associated membranous nephropathy.Clin Kidney J. 2015; 8: 433-439Crossref PubMed Scopus (24) Google a proportion in NELL1-associated MN cases Other described in malignancy-associated MN also in NELL1-associated MN cases include segmental of glomerular capillary and the presence of immune In NELL1-positive patients in the presence of deposits with malignancy. have described in malignancy-associated C. B. et nephropathy and cancer: and of cancer Int. Full Text Full Text PDF PubMed Scopus Google Scholar and or other were present in NELL1-associated MN biopsies. a of MN to be to be malignancy it clinical which are often to in the malignancy and glomerular disease within a A. Porta C. Cosmai L. et al.Glomerular diseases and cancer: evaluation of underlying malignancy.J Nephrol. 2016; 29: 143-152Crossref PubMed Scopus (30) Google Scholar within 2 years of the clinical and of glomerular disease should the cancer is in C. B. et nephropathy and cancer: and of cancer Int. Full Text Full Text PDF PubMed Scopus Google Scholar a of glomerular disease should cancer a is as the same antigen in the and glomerular glomerular diseases with PubMed Scopus (58) Google Scholar These data were present in NELL1-positive that their MN was malignancy The malignancy for years of age of is of the general data Scholar of patients with NELL1-associated MN with malignancy, the is that for the general Although NELL1-associated MN was enriched in patients with malignancy, there were many patients with MN and malignancy an identified the of NELL1 positivity exclude a with MN of unknown from a thorough workup for malignancy. The workup to for in the cancer by in for cancer in cancer by antigen in cancer by and a for of cancer in with a of Other also serum for antigens cancer antigen cancer antigen and A. Porta C. Cosmai L. et al.Glomerular diseases and cancer: evaluation of underlying malignancy.J Nephrol. 2016; 29: 143-152Crossref PubMed Scopus (30) Google data are to determine serum of NELL1 with the underlying malignancy. be used to cancer the of NELL1 in malignancy-associated The types of in patients with NELL1-associated MN are common include many including and and it is reported that there is NELL1 in from data in the Protein C. Lee S. et of the cancer PubMed Scopus Google Scholar only a of patients with malignancies to is to determine the for and of malignancy-associated In NELL1-associated MN has histopathologic that aid in the identification of cases, including a segmental to incomplete capillary loop for IgG and subclass staining. of patients with NELL1-associated MN have a of malignancy. antibodies are in are to with and/or underlying malignancy. a workup to for underlying malignancy is in patients with NELL1-associated of MN were identified in data were to a study by and and for the of in were A consecutive series was used to determine the of NELL1 in cases of This included a of membranous cases, of which 349 had tissue available for staining (Supplementary Figure This was to determine the frequency of NELL1-associated MN MN and membranous lupus Additional NELL1 cases, which were were identified from NELL1 staining of renal biopsies with MN in clinical after NELL1 staining was in as as from cases in which is described The proportion of malignancy-associated MN cases in association with each antigen was samples were from the Patients with MN to have serum samples in a The majority of serum samples were the first clinical after and most patients were the of serum identified patients with NELL1-associated MN in by immunofluorescence staining for NELL1 biopsy tissue for which we NELL1-positive patients and used serum samples to for serum by as described renal biopsy were used including and for the renal 2004; PubMed Scopus Google The renal Med. PubMed Google Scholar microscopy samples were stained with and and immunofluorescence were 3 with antibodies to and and for and and a was applied using were with a IgG subclass was performed cases of MN with frozen tissue for evaluation (n = IgG antibodies were from and IgG was performed frozen tissue from renal biopsies. To this, the frozen tissue in from renal biopsies was 4 in and by of tissue in by bead Protein were with of Protein G for 1 with were then 4 with to were from the using mass spectrophotometric were analyzed by to MS using a mass were a to a the same as used in the A was then used to a from to were eluted from the with an and by by analysis using higher of were performed with a MS was performed by with the higher with a of and MS analysis in the The data were against the most the and the using of of data was using The discovery was for the from were used for for each were to for in to were from the data a was performed for each protein using for the 2 a protein was in only 1 a was performed using the as the EXT2, PLA2R, and THSD7A was performed tissue a of 3 antibodies directed against PLA2R and NELL1 were by a secondary which was to with IgG Immunoresearch, A directed against THSD7A was by an secondary (Jackson Immunoresearch, was with positive and negative The was using immunofluorescence was to be positive there was capillary loop staining in the of staining or staining was performed 3 tissue were and antigen was performed by the to The were with a NELL1 and a IgG NELL1 and IgG was performed tissue from patients with PLA2R-positive MN as negative controls were performed to by of NELL1 with IgG was performed with a confocal Grove, A of were analyzed group. were using of that were for was using the colocalization analysis where the of colocalization of Red-X and was in glomeruli. were for each were then used to determine and of colocalization samples from 2 including 1 biopsy and from a with of the and 1 from a with were stained with a patients had concurrent MN the of an malignancy. staining for NELL1 was performed 4 of tissue the with a IgG Immunoresearch, was used as the secondary NELL1 protein MN, was to and to The were with bovine serum in and then with in bovine serum in for 2 The were with and then with in bovine serum in for 1 The were in with a with and using The was used as a positive A positive serum is a of the same provided by the A of NELL1-positive serum samples and PLA2R-positive serum samples were used for the no and for their reported in this was by the and of the of The is the of the and the of the of with Supplementary in epidermal growth factor-like membranous more than a the study by et Neural 1 Membranous to be of performed a of in patients with NELL1-positive membranous nephropathy (MN) compared with MN cases of other antigenic types cases each of phospholipase A2 1 exostosin and phospholipase A2 1 PDF

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