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NELL1 is a target antigen in malignancy-associated membranous nephropathy

Open AccessPublished:August 19, 2020DOI:https://doi.org/10.1016/j.kint.2020.07.039
      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.

      Graphical abstract

      Keywords

      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.
      • Beck J.L.
      Membranous nephropathy and malignancy.
      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%).
      • Lee J.C.
      • Yamauchi H.
      • Hopper Jr., J.
      The association of cancer and the nephrotic syndrome.
      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.
      • Birkeland S.A.
      • Storm H.H.
      Glomerulonephritis and malignancy: a population-based analysis.
      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).
      • Pani A.
      • Porta C.
      • Cosmai L.
      • et al.
      Glomerular diseases and cancer: evaluation of underlying malignancy.
      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.
      • Ronco P.M.
      Paraneoplastic glomerulopathies: new insights into an old entity.
      Other factors characteristic of malignancy-associated MN include phospholipase A2 receptor (PLA2R) negativity and IgG1- and/or IgG2-restricted subclass predominance.
      • Lonnbro-Widgren J.
      • Ebefors K.
      • Molne J.
      • et al.
      Glomerular IgG subclasses in idiopathic and malignancy-associated membranous nephropathy.
      ,
      • Ohtani H.
      • Wakui H.
      • Komatsuda A.
      • et al.
      Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy.
      Neural epidermal growth factor–like 1 (NELL1) is a recently described antigen in MN.
      • Sethi S.
      • Debiec H.
      • Madden B.
      • et al.
      Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.
      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.

      Results

       Mass spectrometry analysis revealed NELL1 as an antigen in MN

      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.
      Figure thumbnail gr1
      Figure 1Ig capture from frozen kidney biopsy followed by mass spectrometry identifies neural epidermal growth factor–like 1 (NELL1) as a target antigen. The volcano plot shows NELL1 as a potential protein of interest, with a high fold-change and low P value compared to other protein candidates.

       NELL1 colocalizes with IgG in subepithelial glomerular immune deposits

      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).

       Frequency data from NELL1 staining in a consecutive case series of MN

      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 primary MN cases. Of the 349 total cases, 181 were PLA2R positive (51.9%), 10 were THSD7A positive (2.9%), 33 were EXT2 positive (9.5%), and 6 were NELL1 positive (1.7%). Of the 266 primary MN cases only, 180 were PLA2R positive (67.7%), 7 were THSD7A positive (2.6%), 6 were EXT2 positive (2.3%), and 5 were NELL1 positive (1.9%). Of the 83 class V membranous lupus nephritis cases, 1 was PLA2R positive (1.2%), 3 were THSD7A positive (3.6%), 27 were EXT2 positive (32.5%), and 1 was NELL1 positive (1.2%). As there were too small of a number of cases from the 9-month consecutive case series to identify potential clinical or histologic associations of NELL1-associated MN, NELL1-associated MN cases were identified from over the course of 5 years of MN cases. In total, 91 NELL1-associated MN cases were identified.

       Characteristics of NELL1-associated MN (91 patients)

      On light microscopy, NELL1-associated MN cases showed prominent glomerular capillary loops (Figure 2a). Forty-one of 91 cases (45.1%) showed the formation of spikes or holes on silver stains (Figure 2b), often in a segmental distribution in glomeruli. A segmental (involvement of <50% of glomerular tuft) to incomplete global (segmental loops uninvolved by staining) capillary loop staining pattern was seen in 93.4% of cases of NELL1-associated MN (Figure 2c and d) and in no cases of PLA2R-associated (0 of 181) or THSD7A-associated (0 of 10) MN. Complete (100% tuft involvement) capillary loop staining was seen in 6 of 91 NELL1-associated MN (6.6%) compared with 100% of PLA2R-positive MN and 100% of THSD7A-positive MN control cases (Supplementary Figure S4). The segmental pattern of immune deposits was also seen by the distribution of electron-dense immune-type deposits by electron microscopy, showing sparing of some glomerular capillary loops (Figure 2e), with other loops containing electron-dense immune-type deposits (Figure 2f).
      Figure thumbnail gr2
      Figure 2Neural epidermal growth factor–like 1–associated membranous nephropathy shows unique histopathologic features. (a) Glomerulus on periodic acid–Schiff stain showing prominent glomerular capillary loops. (b) Spikes and holes along the glomerular capillary loops seen on silver stain. (c) Immunofluorescence microscopy demonstrating segmental IgG staining in glomeruli. (d) Immunofluorescence microscopy demonstrating incomplete global IgG staining in glomeruli. (c,d) The yellow arrow shows segments of granular capillary loop immunostaining. (ad) Bar = 20 μm. (e) Electron microscopy showing capillary loop sparing of electron-dense immune-type deposits. (f) Electron microscopy showing electron-dense deposits along the glomerular capillary loops. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
      Biopsies of patients with NELL1-associated MN showed no significant difference in IgA positivity (7.7% NELL1 vs. 10% PLA2R; P = .52), IgM positivity (9.9% NELL1 vs. 15.5% PLA2R; P = .26), and C1q positivity (0% NELL1 vs. 2% PLA2R; P = .30) and lacked a full house immunofluorescence pattern with the expression of IgA, IgG, IgM, C3, and C1q (0% NELL1 vs. <1% PLA2R; P = 1.0) as compared with PLA2R-positive MN (Table 1). C3 positivity was less common in NELL1-associated MN than in PLA2R-positive MN (78.0% NELL1 vs. 91% PLA2R; Fisher exact test, P = .002). There was no significant extraglomerular staining in cases of NELL1-associated MN, with no cases demonstrating deposits along tubular basement membranes, Bowman’s capsule, or vessels. EXT2-positive MN cases had a higher frequency of IgA, IgM, C3, and C1q, full house immunofluorescence, and the presence of mesangial electron-dense deposits than did NELL1-associated, PLA2R-positive, or THSD7A-positive MN cases. A summary of the histopathologic features is given in Table 1.
      Table 1Histopathologic parameters of kidney biopsies with NELL1-associated membranous nephropathy compared with PLA2R-associated, THSD7A-associated, and EXT2-associated membranous nephropathy
      ParameterNELL1-positive (n = 91)PLA2R-positive (n = 181)THSD7A-positive (n = 10)EXT2-positive (n = 33)P (Fisher exact test NELL1 vs. PLA2R)
      Incomplete global capillary loop (>50%)44 of 91 (48.3)0 of 181 (0)0 of 10 (0)0 of 33 (0)<0.0001
      Pure segmental (<50%)41 of 91 (45.0)0 of 181 (0)0 of 10 (0)0 of 33 (0)<0.0001
      Segmental or incomplete capillary loop staining85 of 91 (93.4)0 of 181 (0)0 of 10 (0)0 of 33 (0)<0.0001
      IgA positivity7 of 91 (7.7)19 of 181 (10)4 of 10 (40)12 of 33 (36)0.52
      IgG positivity91 of 91 (100)181 of 181 (100)10 of 10 (100)33 of 33 (100)N/A
      IgM positivity9 of 91 (9.9)28 of 181 (15.5)1 of 10 (10)12 of 33 (36)0.26
      C3 positivity71 of 91 (78.0)166 of 181 (91)6 of 10 (80)31 of 33 (94)0.002
      C1q positivity0 of 91 (0)4 of 181 (2)1 of 10 (10)12 of 33 (36)0.30
      Full-house immunofluorescence0 of 91 (0)1 of 181 (<1)0 of 10 (0)5 of 33 (15)1.00
      Mesangial deposits21 of 88
      Mesangial and subendothelial electron-dense deposits are assessed from 88 total cases instead of 91 total cases, as 3 of 91 cases did not have a glomerulus available for evaluation by electron microscopy.
      (23.9)
      19 of 181 (10)4 of 10 (40)26 of 33 (79)0.01
      Subendothelial deposits0 of 88
      Mesangial and subendothelial electron-dense deposits are assessed from 88 total cases instead of 91 total cases, as 3 of 91 cases did not have a glomerulus available for evaluation by electron microscopy.
      (0)
      2 of 181 (1)1 of 10 (10)6 of 33 (18)1.00
      EXT2, exostosin-2; N/A, not applicable; NELL1, nerve epidermal growth factor-like 1; PLA2R, phospholipase A2 receptor; THSD7A, thrombospondin type-1 domain-containing 7A.
      Data include frequency (% total), unless otherwise noted.
      a Mesangial and subendothelial electron-dense deposits are assessed from 88 total cases instead of 91 total cases, as 3 of 91 cases did not have a glomerulus available for evaluation by electron microscopy.
      IgG subclass staining was performed on 67 cases with residual frozen tissue available for testing (Table 2). There was IgG1 staining in all cases of NELL1-associated MN (67 of 67 cases) (Figure 3; Supplementary Figure S5), and 49 of 67 cases were IgG1 dominant (73.1%). Eighteen of 67 cases (26.9%) had IgG1 restriction. Concurrent IgG2 staining was present in 25 of 67 cases (37.3%), and concurrent IgG4 staining was present in 36 of 67 cases (53.7%). Three of 67 cases (4.5%) were IgG1 and IgG2 codominant, and 12 of 67 cases (17.9%) were IgG1 and IgG4 codominant. Therefore, 95.5% of NELL1-associated MN cases had dominant or codominant IgG1 subclass staining (Figure 3). There were 3 of 67 cases with IgG1, IgG2, and IgG3 staining (4.5%) without IgG4, a pattern seen in autoimmune disease.
      Table 2IgG subclass staining in NELL1-associated MN
      Staining intensity trace or greater is included.
      IgG subclassNumber of casesPercentage of total
      IgG167 of 67100
      IgG225 of 6737.3
      IgG310 of 6714.9
      IgG436 of 6753.7
      IgG1-dominant49 of 6773.1
      IgG2-dominant0 of 670
      IgG3-dominant0 of 670
      IgG4-dominant3 of 674.5
      IgG1/IgG2-codominant3 of 674.5
      IgG1/IgG4-codominant12 of 6717.9
      IgG1/IgG2/IgG3-positive3 of 674.5
      IgG1 restriction18 of 6726.9
      MN, membranous nephropathy; NELL1, neural epidermal growth factor–like 1.
      a Staining intensity trace or greater is included.
      Figure thumbnail gr3
      Figure 3Neural epidermal growth factor–like 1–positive membranous nephropathy cases show IgG1 staining (with variable IgG2 and IgG4 expression). (a) IgG1, (b) IgG2, (c) IgG3, and (d) IgG4. (ad) Bars = 20 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
      Electron microscopy was available for 88 of 91 cases of NELL1-associated MN. A majority of cases showed severe podocyte foot process effacement (>50%) (69 of 88 cases [78.4%]), with 11 of 88 cases (12.5%) showing moderate podocyte foot process effacement (25%–50%), and 8 of 88 cases (9.1%) showing mild podocyte foot process effacement (<25%). NELL1-associated MN cases had a higher incidence of mesangial deposits (21 of 88 cases for NELL1 [23.9%] vs. 19 of 181 cases for PLA2R [10%]; Fisher exact test, P = .01) and no subendothelial deposits (0 of 88 cases [0%] vs. 2 of 181 cases [1%]; P = 1.0) (Table 1).

       Clinical associations with NELL1-associated MN

      Patients with NELL1-associated MN were older than patients with PLA2R-positive MN (mean age, 66.8 ± 10.8 years NELL1 vs. 56.4 ± 13.9 years PLA2R), patients with THSD7A-associated MN (mean age, 45.1 ± 16.3 years), and EXT-associated MN (mean age, 39.6 ± 16.1 years). Patients with NELL1-associated MN with malignancy were significantly older than patients without malignancy (71.0 ± 8.6 years vs. 65.0 ± 10.5 years; Student t test, P = .01). There was a slight male predominance (58.2%). Approximately a quarter of NELL1-associated MN cases had a history of diabetes mellitus (22 of 91 [24.2%]); however, only 9% had changes in diabetic nephropathy on biopsy. Twenty of 91 patients with NELL1-associated MN (22.0%) had acute tubular injury on biopsy, and 2 of 91 (2.2%) showed concurrent acute interstitial nephritis. Additional disease associations comparing PLA2R-, THSD7A-, EXT2-, and NELL1-positive MN are provided in Supplementary Table S1.
      Two cases of NELL1-associated MN had a history of Crohn’s disease. To further explore this, we stained all cases of PLA2R-negative and THSD7A-negative MN in patients with a history of Crohn’s disease, ulcerative colitis, or inflammatory bowel disease from our database and found that 2 of 8 cases (25%) were NELL1 positive. Although NELL1 was identified as a gene of interest in Crohn’s disease in a genome-wide association study,
      • Franke A.
      • Hampe J.
      • Rosenstiel P.
      • et al.
      Systematic association mapping identifies NELL1 as a novel IBD disease gene.
      whether this represents a potential secondary etiology for the development of NELL1-associated MN in a subset of patients is unclear.
      Five years of idiopathic MN biopsies from our database were studied to identify malignancy-associated MN cases. One hundred eleven malignancy-associated MN cases were identified, of which 35 were PLA2R positive, 4 were THSD7A positive, and 30 were NELL1 positive (Table 3). Given the much higher prevalence of PLA2R-positive MN than of NELL1-positive MN, patients with cancer are more likely to have PLA2R-positive MN. However, the prevalence of malignancy is much higher in patients with NELL1-positive MN than in those with other types of MN. For example, 33% of NELL1-positive patients had malignancy (30 of 91) whereas only 4.2% of PLA2R-positive patients (35 of 829) and 10.8% of THSD7A-positive patients had concurrent malignancy (4 of 37). Two THSD7A-positive patients and 4 NELL1-positive cases had 2 concurrent primary tumors (Table 3). The associated antigens for malignancy-associated MN were unknown in 42 patients, of whom 3 had 2 primary tumors (Supplementary Figure S6).
      Table 3Increased prevalence of malignancy in NELL1-associated MN compared with PLA2R-associated MN, THSD7A-associated MN, and MN due to unknown antigens
      The types and frequencies of tumors seen in patients with malignancy-associated MN are shown.
      Type of neoplasmPLA2R-positive (n = 35 of 829)THSD7A-positive (n = 4 of 37)NELL1-positive (n = 30 of 91)Unknown antigen (n = 42 of 421)
      Prostate adenocarcinoma103610
      Breast carcinoma12158
      Gastric carcinoma0012
      Soft-tissue tumor0010
      Glioma0010
      Colon adenocarcinoma1016
      Lung carcinoma1034
      Bladder carcinoma5020
      Renal cell carcinoma2135
      Pancreatic carcinoma0001
      Hepatocellular carcinoma0001
      Thyroid carcinoma0012
      Ovarian carcinoma0010
      Uterine carcinoma0001
      Cervical carcinoma1010
      Testicular carcinoma1000
      Skin (melanoma, BCC, SCC)2041
      SCC (head/neck)0112
      Laryngeal carcinoma0001
      Nasopharyngeal carcinoma0010
      Thymoma0010
      Lymphoma0011
      Total356
      Although 4 patients in the THSD7A-positive cohort had a history of malignancy, the number of tumors is >4 because of 2 patients with 2 primary tumors.
      34
      Although 30 patients in the NELL1-positive cohort had a history of malignancy, the number of tumors is >30 because of 4 patients with 2 primary tumors.
      45
      Forty-two patients with MN of unknown type had a history of malignancy, and the number of tumors is >42 because of 3 patients with 2 primary tumors.
      BCC, basal cell carcinoma; MN, membranous nephropathy; NELL1, neural epidermal growth factor–like 1; PLA2R, phospholipase A2 receptor; SCC, squamous cell carcinoma; THSD7A, thrombospondin type-1 domain-containing 7A.
      a The types and frequencies of tumors seen in patients with malignancy-associated MN are shown.
      b Although 4 patients in the THSD7A-positive cohort had a history of malignancy, the number of tumors is >4 because of 2 patients with 2 primary tumors.
      c Although 30 patients in the NELL1-positive cohort had a history of malignancy, the number of tumors is >30 because of 4 patients with 2 primary tumors.
      d Forty-two patients with MN of unknown type had a history of malignancy, and the number of tumors is >42 because of 3 patients with 2 primary tumors.
      Of note, the epidemiology of MN changed within this 5-year time period, where PLA2R-positive MN made up 87% of cases of MN at our institution in 2014, but only 42% in 2019. We attribute this to serum-based approaches to the diagnosis of primary MN,
      • De Vriese A.S.
      • Glassock R.J.
      • Nath K.A.
      • et al.
      A proposal for a serology-based approach to membranous nephropathy.
      with anti-PLA2R and anti-THSD7A assays now widely available for screening of patients presenting with nephrotic syndrome.
      A majority of patients with NELL1-associated MN with a history of malignancy had concurrent proteinuria and ongoing malignancy (n = 19 of 30); 8 patients had the cancer identified before MN; 1 patient had malignancy identified in a workup for causes of secondary MN; and in 2 patients, the underlying malignancy preceded the diagnosis of MN but the time of diagnosis was unknown (Supplementary Table S2). Seven of these patients had metastatic disease at the time of diagnosis. We excluded patients in whom the diagnosis of malignancy was remote compared with the diagnosis of MN or in complete remission (n = 4 of 34 NELL1-positive patients had a history of malignancy; 30 patients in our cohort had active malignancy). The timing between the diagnosis of cancer and MN in our cohort (when known) is summarized in Supplementary Table S2.
      Serum creatinine at the time of biopsy and quantitative 24-hour proteinuria values were similar in patients with NELL1-associated MN and those with PLA2R-, THSD7A-, or EXT-associated MN (serum creatinine: PLA2R 1.93 ± 2.19 mg/dl, THSD7A 1.85 ± 2.34 mg/dl, NELL1 1.33 ± 0.86 mg/dl [data available for 73 of 91 patients]; 24-hour proteinuria: PLA2R 8.03 ± 5.09 g, THSD7A 6.68 ± 4.76 g, NELL1 6.6 ± 4.1 g [for which data were available for 59 of 91 patients]). A majority of patients in all groups had normal renal function at the time of biopsy, with the variation due to outliers with concurrent acute kidney injury. A majority of patients with NELL1-associated MN (71.2%) had nephrotic range proteinuria (≥3.5 g) at the time of biopsy (corresponding to 42 of 59 patients with available quantitative proteinuria data). Depressed albumin levels were seen in 74.5% of patients (38 of 51 patients with serum albumin values available with <3.5 g/dl albumin; mean albumin, 2.84 ± 0.95 g/dl).

       Tumor staining for NELL1

      Tissue of the associated tumor was available from 2 patients with NELL1-associated MN. In 1 patient with NELL1-associated MN and concurrent invasive ductal carcinoma of the breast, NELL1 positivity by immunofluorescence and immunohistochemical staining was seen in the patient’s breast biopsy specimen (Supplementary Figure S7A and B) and mastectomy specimen (Supplementary Figure S7C and D). A patient with active follicular lymphoma also demonstrated NELL1 staining on an excisional lymph node biopsy (Supplementary Figure S7E and F).

       NELL1 antibodies are detected in sera from patients with NELL1-associated MN

      Twenty-eight patients with NELL1-associated MN had blood samples available for testing serum reactivity against the NELL1 antigen. Twenty of 28 patients with NELL1-associated MN (71.4%) had serum reactivity to NELL1 recombinant protein under nonreducing conditions (representing Western blots in Supplementary Figure S8). No control sera from PLA2R-positive MN cases showed reactivity (0 of 28 patients). Of note, 3 of 8 NELL1-positive patients without serum reactivity were in clinical remission, with a >50% reduction in proteinuria at the time of serum collection compared with the time of biopsy.

       Clinical follow-up

      Clinical follow-up with treatment response (evaluating remission of MN) and laboratory data were available from 59 of 91 patients (64.8%). In addition to these 59 patients, 8 of 91 patients in our cohort were not on active treatment at follow-up as they were deceased or on dialysis (8 of 91 [12%]) and 6 of 91 (6.6%) did not return for clinical follow-up after their biopsy. The mean clinical follow-up period was 10.4 ± 13.5 months. The median proteinuria (± interquartile range) at follow-up was 1.9 ± 4.1 g. The median change in proteinuria from the time of biopsy was 5.4 ± 6.6 g.
      Twenty of 59 patients (34%) were in complete remission, with <300 mg of proteinuria per day. Sixteen of 59 patients (27%) were in partial remission, with 300 to 3500 mg of proteinuria per day or a >50% reduction in proteinuria from the time of biopsy. Twenty-three of 59 patients (39%) had no remission, with <50% reduction in proteinuria and >3.5 g of proteinuria per day. A majority of patients had stable renal function at follow-up (median creatinine, 1.1 ± 0.56 mg/dl).
      Comparisons of the effects of various treatments were between patients with available data (n = 59), and sample medians were provided. For patients on renin-angiotensin system blockade (with angiotensin-converting enzyme inhibitor and/or angiotensin receptor blockade therapy; n = 32), the median proteinuria at the time of biopsy was 4.9 ± 4.7 g, the median proteinuria at follow-up was 1.9 ± 4.0 g, and the median follow-up creatinine was 1.1 ± 0.4 mg/dl. In calcineurin inhibitor–treated patients (n = 8), the median proteinuria at the time of biopsy was 10 ± 9.1 g, the median proteinuria at follow-up was 6.4 ± 10.4 g, and the median follow-up creatinine was 1.8 ± 3.0 mg/dl. In patients treated with cyclophosphamide (n = 3), the median proteinuria at the time of biopsy was 9.6 g, the median proteinuria at follow-up was 3.9 g, and the median follow-up creatinine was 1.4 mg/dl (interquartile range not provided because of low n). In patients treated with mycophenolate and steroid therapy (n = 3), the median proteinuria at the time of biopsy was 8 g, the median proteinuria at follow-up was 0.1 g, and the median creatinine at follow-up was 0.9 mg/dl (interquartile range not provided because of low n). One patient was treated with rituximab, who presented with 10 g of proteinuria and had >10 g of proteinuria at follow-up and 1.0 mg/dl of creatinine at follow-up.
      For patients undergoing active treatment of an underlying malignancy and not on concurrent immunosuppressive therapy (n = 12), the median proteinuria at presentation was 5 ± 7.4 g, the median proteinuria at follow-up was 0.1 ± 2.1 g, and the median follow-up creatinine was 1.0 ± 0.9 mg/dl. Because of the small number of patients on immunosuppressive therapy (n = 15), an optimal treatment of NELL1-associated MN could not be ascertained; however, patients with successful treatment of an underlying malignancy showed good response to therapy (with 9 of 12 patients treated for malignancy without concurrent immunosuppression in complete or partial remission at follow-up). The treatment and follow-up data after therapy is included in Supplementary Table S3.

       Limitations

      Our study has several limitations. Complete clinical follow-up was not available for all patients. As patients with malignancy-associated MN can have cancer identified during a workup after the diagnosis of their glomerular disease up to 2 to 4 years after MN and we did not have longitudinal follow-up of all patients for ≥4 years, our data likely underestimate the true frequency of malignancy in patients with MN.

      Discussion

      NELL1 is a 140-kDa modular glycoprotein, which is expressed in low levels in adult tissues, but is active during development where it is essential for intramembranous and endochondral ossification. It shows sequence similarity to thrombospondin proteins, and its structure consists of 1 N-terminal laminin G–like domain, 3 von Willebrand factor type C (VWF-C) domains, 1 thrombospondin type-1 domain, and 5 tandem epidermal growth factor domains.
      • Kuroda S.
      • Oyasu M.
      • Kawakami M.
      • et al.
      Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.
      NELL1, unlike PLA2R or THSD7A, does not contain a transmembrane domain and has a secretory signal sequence.
      Sethi et al. recently showed that NELL1-positive MN was a specific subtype of MN and was the responsible antigen in up to 16% of PLA2R-negative primary MN cases.
      • Sethi S.
      • Debiec H.
      • Madden B.
      • et al.
      Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.
      We identified NELL1-associated MN in 3.8% of primary MN cases that were negative for PLA2R and THSD7A. An association with malignancy was higher in our cohort (33%) compared with their cohort malignancy association of 11.7%.
      The histopathology of NELL1-associated MN was unique compared with other forms of MN. This includes segmental to incomplete global IgG staining, IgG1 subclass positivity, and lack of staining for other immune reactants (IgA, IgM, and C1q). Many of the histopathologic features observed in NELL1-associated MN have previously been described in reports of malignancy-associated MN in the literature. These include IgG1 and IgG2 subclass specificity, while primary PLA2R-positive MN cases more frequently had IgG4-dominant deposits (65%) and malignancy-associated MN cases had a reduced frequency of IgG4 dominance (31%
      • Lonnbro-Widgren J.
      • Ebefors K.
      • Molne J.
      • et al.
      Glomerular IgG subclasses in idiopathic and malignancy-associated membranous nephropathy.
      ), a similar proportion in NELL1-associated MN cases (22.4%). Other features described in malignancy-associated MN also seen in NELL1-associated MN cases include segmental sparing of glomerular capillary loops and the presence of mesangial immune deposits. In NELL1-positive patients in our cohort, the presence of mesangial deposits did not correlate with malignancy. Previous reports have described endocapillary hypercellularity in malignancy-associated MN,
      • Lefaucheur C.
      • Stengel B.
      • Nochy D.
      • et al.
      Membranous nephropathy and cancer: epidemiologic evidence and determinants of high-risk cancer association.
      and endocapillary hypercellularity or other proliferative changes were not present in NELL1-associated MN biopsies.
      For a case of MN to be considered to be malignancy associated, it must fulfill certain clinical criteria, which are often difficult to establish clinically in individual cases. First, the patient’s malignancy and glomerular disease must occur within a similar time frame,
      • Pani A.
      • Porta C.
      • Cosmai L.
      • et al.
      Glomerular diseases and cancer: evaluation of underlying malignancy.
      generally within 2 years of one another. Second, the clinical and histologic remission of glomerular disease should occur once the cancer is in remission.
      • Lefaucheur C.
      • Stengel B.
      • Nochy D.
      • et al.
      Membranous nephropathy and cancer: epidemiologic evidence and determinants of high-risk cancer association.
      Third, a relapse of glomerular disease should occur upon cancer recurrence. Fourth, a pathophysiologic link is helpful, such as the same antigen expressed in the tumor and glomerular disease.
      • Cambier J.F.
      • Ronco P.
      Onco-nephrology: glomerular diseases with cancer.
      These temporal data were present in selected NELL1-positive patients, supporting that their MN was malignancy associated.
      The age-adjusted malignancy incidence rate for 65 years of age of both sexes is 1.9% of the general population.

      National Cancer Institute, Interactive tools, maps, & graphs: data query tools: SEER Explorer. Available at: https://seer.cancer.gov/statistics/interactive.html. Accessed December 17, 2020.

      For 30 of 91 patients with NELL1-associated MN with malignancy, the standardized incidence ratio is 17.4 times that expected for the general population. Although NELL1-associated MN was enriched in patients with malignancy, there were many patients with MN and malignancy without an identified autoantigen. Therefore, the lack of NELL1 positivity would not exclude a patient with MN of unknown type from undergoing a thorough workup for malignancy. The recommended workup to screen for neoplasia in the United States includes breast cancer screening by mammography in women, pap testing for cervical cancer in women, prostate cancer screening by prostate-specific antigen testing in men, colon cancer screening by colonoscopy, and a low-dose computed tomography scan for screening of lung cancer in individuals with a history of smoking. Other guidelines also recommend serum testing for tumor-associated antigens (including cancer antigen 125, carcinoembryonic antigen, cancer antigen 19-9, and alpha-fetoprotein
      • Pani A.
      • Porta C.
      • Cosmai L.
      • et al.
      Glomerular diseases and cancer: evaluation of underlying malignancy.
      ). Further data are needed to determine whether serum levels of NELL1 correlate with the patient’s underlying malignancy. If so, serological testing could be used to monitor cancer recurrence.
      Questions remain regarding the pathophysiology of NELL1 in malignancy-associated MN. The types of tumors seen in patients with NELL1-associated MN are common (to include many carcinomas including prostate cancer, lung cancer, and breast cancer), and it is reported that there is high NELL1 expression in these tumors from data in the Human Protein Atlas.
      • Uhlen M.
      • Zhang C.
      • Lee S.
      • et al.
      A pathology atlas of the human cancer transcriptome.
      However, only a small percentage of patients with these malignancies will go on to develop MN. Therefore, further work is required to determine the pathophysiologic mechanism for anti-NELL1 antibody production and subsequent development of malignancy-associated MN.
      In summary, NELL1-associated MN has histopathologic features that aid in the identification of cases, including a segmental to incomplete global granular capillary loop pattern for IgG and IgG1 subclass staining. One-third of patients with NELL1-associated MN have a history of malignancy. Anti-NELL1 antibodies are detected in patient sera; however, further studies are required to elucidate whether antibody titers correlate with proteinuria and/or underlying malignancy. Nonetheless, a workup to evaluate for underlying malignancy is recommended in patients with NELL1-associated MN.

      Methods

       Case selection

      Cases of MN were identified in our case archives. All data were collected according to a study protocol approved by Solutions IRB (Little Rock, AR), and all ethical principles and guidelines for the protection of human subjects in research were followed. A 9-month consecutive case series was used to determine the prevalence of NELL1 in all cases of MN. This cohort included a total of 404 membranous cases, of which 349 had residual tissue available for staining (Supplementary Figure S9). This cohort was screened to determine the frequency of NELL1-associated MN among primary MN and membranous lupus nephritis. Additional NELL1 cases, which were nonconsecutive, were identified from NELL1 staining of renal biopsies with MN in our clinical practice after NELL1 staining was validated in our laboratory as well as from screening cases in our biorepository, which is described below. The proportion of malignancy-associated MN cases in association with each antigen was evaluated.
      Serum samples were obtained from the Arkana Laboratories biorepository. Patients with PLA2R-negative MN consented to have serum samples banked in a research protocol. The majority of serum samples were collected at the first clinical follow-up visit after diagnosis, and most patients were not treatment naive at the time of serum collection. We identified patients with NELL1-associated MN in our biorepository by immunofluorescence staining for NELL1 on residual formalin-fixed paraffin-embedded biopsy tissue for which we found 28 NELL1-positive patients and used serum samples to evaluate for serum reactivity by Western blotting as described below.

       Renal biopsy processing techniques

      Standard renal biopsy analytical processing methods were used including light, immunofluorescence, and electron microscopy.
      • Walker P.D.
      • Cavallo T.
      • Bonsib S.M.
      Practice guidelines for the renal biopsy.
      ,
      • Walker P.D.
      The renal biopsy.
      All light microscopy samples were stained with hematoxylin and eosin, Jones methenamine silver, Masson trichrome, and periodic acid–Schiff reagent. All direct immunofluorescence sections were cut at 3 μm; reacted with fluorescein-tagged polyclonal rabbit anti-human antibodies to IgG, IgA, IgM, C3, C1q, fibrinogen, and κ and λ light chains for 30 minutes; and rinsed, and a coverslip was applied using aqueous mounting media. For electron microscopy, thin sections were examined with a JEM-1011 electron microscope (Jeol, Tokyo, Japan).
      IgG subclass typing was performed on cases of MN with residual frozen tissue for evaluation (n = 67). IgG antibodies were all goat polyclonal direct FITC conjugates from Jackson Immunoresearch (IgG1, catalog #115-095-205; IgG2, catalog # 115-095-207; IgG3, catalog # F4641; and IgG4, catalog # F9889).

       Protein G tissue immunoprecipitation

      IgG coimmunoprecipitation was performed on fresh frozen tissue from renal biopsies. To do this, the residual frozen tissue in optimal cutting temperature (OCT) medium from archived renal biopsies was thawed, washed 4 times in phosphate-buffered saline (PBS), and lysed by mechanical disruption of tissue cores in Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA) by bead beating. Protein extracts were incubated with 50 μl of magnetic Dynabeads Protein G (Invitrogen, Thermo Fisher Scientific) at room temperature for 1 hour with shaking. Samples were then washed 4 times with PBS to reduce nonspecific binding interactions. Proteins were digested from the beads using trypsin before mass spectrophotometric analysis.

       MS

      Digested peptides were analyzed by nanoscale liquid chromatography coupled to tandem MS using a Thermo Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a reverse phase trap column (IntegraFrit, New Objective, Littleton, MA) containing 2.5 μm Waters XSelect CSH resin (Waters Corporation, Milford, MA) coupled to a 150 mm × 0.075 mm analytical column containing the same reverse phase resin as used in the trap. A nanoACQUITY UPLC system (Waters Corporation, Milford, MA) was then used to generate a 60-minute gradient from 98:2 to 60:40 buffer A/buffer B ratio (buffer A: 0.1% formic acid, 0.5% acetonitrile; buffer B: 0.1% formic acid, 99.9% acetonitrile). Peptides were eluted from the column with an integrated spray tip (PicoFrit, New Objective) and ionized by electrospray (2.0 kV) followed by MS/MS analysis using higher energy collision–induced dissociation. Survey scans of peptide precursors were performed at 240,000 resolution (at 400 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.6 Th with the quadrupole, higher energy collision–induced dissociation fragmentation with a normalized collision energy of 30 eV, and rapid scan MS analysis in the ion trap. The obtained MS/MS data were searched against the most recent UniProt human database containing both the Swiss-Prot and the TrEMBL entries using MaxQuant (Max Planck Institute of Biochemistry). Visualization of data was done using Scaffold v4.6 (Proteome Software Inc., Portland, OR). The false discovery rate was set at 1% for the peptide-to-spectrum matches. Normalized intensity-based absolute quantification (iBAQ) values from MaxQuant were used for quantitation. iBAQ distributions for each sample were adjusted to control for differences in loading. iBAQ values equal to zero were removed from the data set. For statistical hypothesis testing, a 2-sample t test was performed for each protein using normalized iBAQ values for the 2 groups. If a protein was detected in only 1 group, a 1-sample t test was performed using the smallest detected iBAQ value as the null hypothesis.

       Immunostaining for membranous antigens

      NELL1, EXT2, PLA2R, and THSD7A immunostaining was performed on formalin-fixed paraffin-embedded tissue sections cut at a thickness of 3 μm. Rabbit polyclonal antibodies directed against human EXT2 (Abcam, Cambridge, MA, catalog # AB102843), human PLA2R (Sigma, St. Louis, MO, catalog # HPA012657), and human NELL1 (Thermo Fisher Scientific, catalog # PA5-27958) were followed by a Rhodamine Red X–conjugated goat anti-rabbit secondary antibody, which was solid phase adsorbed to ensure minimal cross-reaction with human IgG (1:100; Jackson Immunoresearch, catalog # 111-295-144). A mouse monoclonal antibody directed against THSD7A (Atlas antibodies/Sigma-Aldrich, St. Louis, MO, catalog # AMAb91234) was followed by an FITC-conjugated goat anti-mouse secondary (Jackson Immunoresearch, catalog # 115-095-205). Each case was run with positive and negative controls. The stain was evaluated using immunofluorescence microscopy. Each stain was judged to be positive if there was granular capillary loop staining in the glomeruli of trace staining or greater.

       Confocal microscopy

      Immunofluorescence staining was performed on 3 μm formalin-fixed paraffin-embedded tissue sections. Formalin-fixed paraffin-embedded sections were deparaffinized, and antigen retrieval was performed by heating the slides to 99 °C. The sections were dual labeled with a rabbit polyclonal NELL1 antibody and a FITC-conjugated anti-human IgG antibody. NELL1 and IgG costaining was performed on tissue sections from patients with PLA2R-positive MN as negative controls. Negative controls were performed to ensure antibody specificity by omitting primary antibodies.
      Colocalization of NELL1 with IgG was performed with a Leica DMi8 confocal microscope (Leica Biosystems, Buffalo Grove, IL). A total of 10 glomeruli were analyzed per group. Confocal images were captured using sequential scanning of Z-stack images that were overlayed for maximal projection. Colocalization was quantified using the colocalization Leica image analysis software (Leica Biosystems), where the percentage of colocalization of Rhodamine Red-X and FITC was determined in glomeruli. Pearson correlation coefficients were determined for each glomerulus. Student t tests were then used to determine differences and percentage of colocalization between groups.

       NELL1 staining of tumor tissue

      Three tumor samples from 2 patients, including 1 biopsy and mastectomy specimen from a patient with invasive ductal carcinoma of the breast, and 1 lymph node resection from a patient with follicular lymphoma were stained with a rabbit polyclonal anti-NELL1 antibody. Both patients had concurrent MN at the time of an active malignancy. Immunohistochemical staining for NELL1 was performed on 4 μm sections of tumor tissue on the Leica BOND platform (Leica BioSystems) at 1:100 primary dilution with a anti-NELL1 rabbit polyclonal antibody (Thermo Fisher Scientific, PA5-27958). Rhodamine Red-X–conjugated goat anti-rabbit IgG (1:100; Jackson Immunoresearch, catalog # 111-295-144) was used as the secondary antibody.

       Western blotting

      NELL1 recombinant protein (R&D Systems, Minneapolis, MN, catalog # 5487-NL) was electrophoresed on NuPAGE 4% to 10% Bis-Tris gels (Invitrogen) at 0.2 μg/lane and transferred to polyvinylidene diflouride membranes under nonreducing conditions. The membranes were blocked with 5% bovine serum albumin solution in PBS containing 0.1% Tween and then incubated with patient sera at 1:50 dilution in 2% bovine serum albumin in PBS for 2 hours at room temperature. The membranes were washed with PBS containing 0.1% Tween and then incubated with anti-human IgG-horseradish peroxidase at 1:1000 dilution in 2% bovine serum albumin in PBS containing 0.1% Tween for 1 hour at room temperature. The membranes were washed in PBS containing 0.1% Tween, with a final wash with PBS alone, and developed using 3,3′-diaminobenzidine substrate. The rabbit polyclonal anti-NELL1 antibody (Thermo Fisher Scientific, PA5-27958) was used as a positive control. A positive serum result is a band of the same molecular weight provided by the anti-NELL1 antibody. A total of 28 NELL1-positive serum samples and 28 PLA2R-positive serum samples were used for evaluation.

      Disclosure

      All the authors declared no competing interests.

      Acknowledgments

      We thank Sudhir Joshi and Lilli Barnum for their technical assistance. Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under award number R43MD014110. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supplementary Material

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      Linked Article

      • Lipoic acid in neural epidermal growth factor-like 1–associated membranous nephropathy: more than a coincidence?
        Kidney InternationalVol. 101Issue 2
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          We found the study by Spain et al., “Lipoic Acid Supplementation Associated With Neural Epidermal Growth Factor-Like 1 (NELL1)–Associated Membranous Nephropathy,” to be of great interest.1 We performed a retrospective review of medications in 115 patients with NELL1-positive membranous nephropathy (MN) compared with 200 MN cases of other antigenic types (50 cases each of phospholipase A2 receptor, thrombospondin-type 1 domain containing 7A, exostosin 1/2, and phospholipase A2 receptor/thrombospondin-type 1 domain containing 7A/exostosin/NELL1-negative MN).
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